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GraFit 7 User`s Guide

1. v KglA K4 B A B T Ping pong Lineweaver E Burk A This transformation generates a 0 6 A Lineweaver Burk plot for the fitted data E i o aan 0 0 2 4 1 A Ternary aa ra B 50 Use this equation where a_ ternary O oas complex is formed The equation relating dl e the rate of reaction v to the a oe ce concentration of substrates A and B and fr lA E the constants K K and K is Sl A 0 2 4 6 8 10 A 7 Vax lA B K Kg KglA KalB A B T Ternary Lineweaver Burk This transformation generates a Lineweaver Burk plot for the fitted data E 266 Chapter 22 Equations Provided Enzyme Inhibition Competitive Inhibition In this multidimensional equation rate is monitored versus substrate and inhibitor The equation for competitive inhibition is E pz S Eo kcat S Kin 1 1 K Substrate T Competitive Eadie Hofstee ie Use this to display the Eadie Hofstee plot Es Rate Substrate T Competitive Lineweaver Burk This transformation displays the Lineweaver Burk plot 2 Mixed Inhibition In this most general inhibition equation rate is monitored versus substrate and inhibitor using the equation M Unas S Km 1 52 1 s T Mixed Lineweaver Burk This transformation displays the Lineweaver Burk plot T Mixed Eadie Hofstee Use this to di
2. cccccsscccsssecccesecccceecceseeeceseesesseeeesseeeesees 190 Running the Axis Manager ccsssccccssseccccnsecccensecccenececsuseeeceececseuseessaneeeseas 190 Addme a NS WAI ri 191 Splitting an EXISHMS AS ia 192 IO VIS IAS a 193 Working with MUItIpIe AXES iia id 194 Pods Text and Axis SCale Texte 194 Data ASSOC ALO ll saicect srcecra T E E E E E E 194 POUS a E nS 195 Column a a Bar Carts sicccscnisediccri coed cavcoadscate dan cave cian tess O a 199 TASE CAIR WIZI Ooae T ii 199 Select Data QUES PIBE aa a tias 199 SEG MIRA CINE sherret eare aa ER 201 Select Tak INES AAEE AE TE AET TE Ae 202 5 eo Bar Styl Eana a ana a a a 203 Select Data PONES S 204 SEISCE ION Soria 205 e sececcstcsccncucessccceourdeecenss duc sacedsquuesescccuesussucuwacccscvescdeoaunsee saauesssdoouenai caenstceceneesesonies 207 Wie Histograma Wizard dica 207 Select Data Source PaBl ooccccccccnnococnnncnncnnncnnnnnnnonnnnnnnnnnnnnnnnnnnnnnnnonannnnnnnnnnnnnannss 207 A A 208 Select TICK Style Sina OT a E T T T 209 SEIE Bale GUY IC a ESS A 210 Embedding and Linking sessssesesecessssesecsececscsesesececessesesececscsesesececscsesesececessesesececesseseeese 211 What is Embedding and LinkiNg coooccccccocnnccnocnnnnononnnnnnonnnnnnononnnnnnnnoncnnonanonnnns 211 Original Cut and Paste Modeleren anran rE NEE EE ENE aS 211 Embedded Oberta adi 211 Linked OD G CUS aa a 212 Usine EmbDedalme and EMKE td 212 Comparison of
3. The Index column selection specifies the column on which the sort order is based In addition to this column any of the other columns can be included in the sort by checking the box to the left of the column name Data Table Appearance Colors and Fonts The colors used to display the items in the data table and the font that is used to draw the table are set using the Preferences command on the Table tab Number Format The manner in which numbers are formatted for display can be set on a column by column basis The Column Format command on the Table tab is used to control the displayed numerical precision This command applies to all columns that fall within the current selection 27 The Table gt Sort Column s command in legacy mode 28 The Table Preferences command in legacy mode 2 The Table Column Format command in legacy mode 63 GraFit User s Guide 64 Column Format Number Format L Decimal places Max digits E Thousands separator E Scientific format Use scientific format for small values Column Format Trailing zeros in fraction Show O s dialog box Save As Defaults Restore Defaults Specification Decimal places Max digits Thousands separator Scientific format Use scientific format for small values Meaning Enter the number of decimal places to be displayed for numbers Note To ensure that all decimal places
4. Data Association Any dataset that is added to a graph can be associated with one or all axes When using multiple axes to display data that have distinct ranges it is usual to associate each dataset with one specific axis For split axes it is usual to associate all datasets with each axis The data association for an axis is set from the Axis Association Page found by double clicking on The Edit gt Properties command in legacy mode The Edit Hide Object command in legacy mode 194 Chapter 15 Using Multiple Graph Axes the appropriate Data Curve Properties region of the data legend see figure above Further information about modifying data properties is found in Chapter 14 Axis Scaling and Sizing Each of the multiple axes has separate scaling size and tick settings These are modified from the Scale or Ticks pages of the graph properties To access these select the graph and choose the Properties command or double click on the main graph region For more detailed information about setting these properties see Chapter 14 Scaling AY Graph Properties Seale check box for autoscale Y ads selector Type Linear Y axis 2 ha V Start D Axis extent tt Finish 110 g Maio 100 El Major increment Fa Minor increment Drawing options Fa X ads T ao position Draw X axis and ticks Use the Axis selector to choose which axis is to be scaled then set the scale as appropri
5. strColumnName The new name of the column sub GF SetObJectDirty ByRef SstrCaleshe t As String ByRef strGFObject As String Show an embedded GraFit object to indicate it is dirty strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object sub GF S cob ectClean ByRert strCalesheet As String ByRef strGFObject As String Show an embedded GraFit object to indicate it is clean strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object 321 GraFit User s Guide 322 Public sub GF SetColummAssocietedDat ByRef strCalcshest As String ByRef strGFObject As String ByRef strGFData As String ByVal iColumn As Integer ByVal datal As Double ByVal data2 As Double ByVal data3 As Double ByVal data4 As Double Sets the data associated with a data colmn in an embedded GraFit object strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object strGFData Name of the data table item within the GraFit object Column The number of the data column start at 1 datal Data value 1 data2 Data value 2 data3 Data value 3 data4 Data value 4 Public Sub GF SetColumnDataValue ByRef strCalcSheet As String ByRef strGFObject As String ByRef strGFData As String ByVal col As Long ByVal row As Long _ ByRef dataValue As Variant Sets a data value within a data
6. a 2 7 40 E E 400 gt 200 Errors Single Exponential 0 e rrr aed E 1600 1400 sea Single xponential S een g 1000 i Exponential 800 600 400 200 Only the Y Data Contain Errors Only the dependent variable which by convention is always the y variable y is subject to error the other x is known exactly This requirement should be satisfied by suitable experimental design the x data values often comprise time or concentration values which should be known with precision The Errors are Normally Distributed The errors in the y data values should follow a normal distribution function i e if sufficient replicates were taken they would have a Gaussian spread about the true y value Two common factors that invalidate this assumption are the presence of i systematic errors and i outliers data points that are wildly inaccurate usually due to operator or machine failure Systematic errors invalidate any data analysis and should be removed by correct experimental design Outliers can be eliminated by use of robust techniques as will be described later in this chapter The Correct Weighting is used An explanation of what weighting of data in regression analysis is why it is incorporated into all such analysis including the subjective regression performed by drawing the best line using a ruler and how the appropriate weighting can be determined are
7. Setting Description Thousands Check this option to separate groups of digits by a separator separator Typically digits are grouped into threes and separated by a comma However the grouping and the separator used depend on values set by in the Options command Number Format pane found on the View tab Scientific format Check this option to display numbers using scientific format For example 1 234e 003 rather than 1234 Power format Check this option to display numbers using a power representation For example 1 234 x 10 rather than 1234 Trailing zeros in This controls the way that trailing zeros are displayed in a fraction fraction Some examples of different formatting are shown below all have Decimal places set to 4 and Max digits set to 10 Effect Settings Effect Settings 0 01 Omit 0 s le 2 Omit 0 s scientific format 0 005 De d J 0 0100 Show 0 s 107 Omit 0 s power format 0 00s Sl 0 0000 d Text These settings control the text display Setting Description Alignment The scale text can be aligned to the left right or centrally Orientation Select the orientation of the text The following orientations are available The View gt Options command in legacy mode 173 GraFit User s Guide Setting Description Format string Check this option to embed the numeric value within a text string The number is substituted for any occurrences of 51 within the entered text Values The S
8. which can be opened using the File Open command Two columns are present in the data table pH and Chemical Shift No other workspace items are present Load the Equation The first action to take is to load the correct equation Use the Add Stock Item command on the Home tab select the pH tab and choose the pKa Single equation definition item Add a Non linear Fit Item to the Workspace Use the Add gt Non linear Fit command to add a non linear fit item to the GraFit workspace Fill out the Non linear Fit Item Settings Complete the Settings page as shown below Ea Non linear Fit Fitting Criteria Results Number Format Fonts Indents Settings Constants Estimatrix Equation pita Single Y Weighting Simple a Robust Data table Data Data layout x y E Limit x range Y data column y Set up Initial Estimates The pKa Single equation is one for which initial estimates for the parameter values are difficult to provide automatically Therefore it is necessary to enter these initial estimates explicitly For equations that require initial estimates it is essential to set these from the Estimatrix page Click on the Estimatrix tab then on the Interactive Estimator button 2 The File Add Stock Item command in legacy mode Chapter 21 Worked Data Fitting Examples Fitting Criteria Results Number Format Fonts Indents Settings Constants Estimatrix Y Link to table Paramete
9. Apart from the considerations above fitting data that have multiple independent variables is no different from fitting standard equations A sample set of data that has been fitted in this way can be found in the Examples data subdirectory and is named Multiple rate 225 GraFit User s Guide Plotting Results from Multiple Regression 226 The data that form the basis for multiple regression have three or more dimensions To represent such data would strictly speaking require a multidimensional plot which is not available However the usual representation for such data is to plot multiple curves representing the raw data as collected on the same graph This is particularly simple for the situation noted as Case 1 above Choosing to create a graph to display the non linear fit will automatically generate a suitable plot In the case of the sample data set the fitted curve is shown below The file that generated this curve can be found in the Samples sub directory of your My GraFit Data directory named Multiple rate 0 6 o 0 4 2 4 Q O uN O LT 02 0 0 2 4 6 8 10 Time CHAPTER 20 Theory To make best use of GraFit it is helpful to know some of the theory behind regression analysis Chapter 19 provided a general overview this section describes more of the theoretical background Regression Analysis Regression analysis is a technique used to calculate the best fit line or cur
10. Column Charts are categorized by having a numeric range on the y axis and named categories on the x axis Some of the types of chart that can be created are illustrated below 60 800 000 Direct lt 600 000 via 2 40 E Internet y n 3 S S u 400 000 e 20 200 000 0 MPC 10234 MPC 12300 MPC 12308 BGF 1034 1994 1995 1996 1997 Compound Year Simple comparison of a set of parameter Summed area plot 135 GraFit User s Guide values 25 10 12 20 Y 8 10 O 8 E f a 6 E 7 h T 10 A amb YO bs a Y Y y A IA Y Y eI MIMI IPI ILIBIBID Et amp S 5 AB AA IAN HABANA Lo l RIA A B C D E Sf S g OOOO mes s Lee SS EOS ce Month A Low High column chart A chart with different y axis scales for the two data sets plotted Bar Chart Bar Charts are categorized by having a numeric range on the x axis and named categories on the y axis and have the same range of styles available as column charts Some of the types of chart that can be created are illustrated below MPC 10234 A B 2 MPC 12300 YW 2 3 C E O O MPC 12308 D BGF 1034 E 0 20 40 60 0 2 4 6 8 10 12 IC Values nM Range Comparison of values using a bar chart A Low High bar chart Histogram Histograms have numeric ranges on both x and y axes the x axis represents value ranges and the y axis shows the number of occurrences within the range Some histogram styles are shown below Note Despite the similar appearance a histogram is funda
11. Customize Add Ins ScreenTip style Show feature descriptions in ScreenTips lz la Open from Office Live gt Trust Center Create lists for use in sorts and fill sequences Edit Custom Lists Resources When creating new workbooks Save to Office Live Use this font Body Font 2 dep Print gt Font size 11 7 Default view for new sheets Normal View 7 A Prepare Include this many sheets 3 gt Send Personalize your copy of Microsoft Office User name Robin Leatherbarrow r4 Pyblish Choose the languages you want to use with Microsoft Office First select Excel Options then ensure Show Developer tab is checked 2 Launch Visual Basic In Excel 2007 select the Developer tab and click Launch Visual Basic Book1 Microsoft Excel Developer l2 3 Map Properties A Import E g Expansion Packs lt 7 Export EAN Refresh Data a ez Sf Properties QH View Code Insert Design Mode Run Dialog In Excel 2003 use the Tools Macro gt Visual Basic Editor command 3 Ensure the GraFit Type Library is Selected In the Visual Basic Editor select the Tools References command to open the References dialog box 316 Chapter 24 Passing Data Between Applications References VBAProject Available References Visual Basic For Applications Microsoft Excel 12 0 Object Libr
12. Enzyme kinetics plot The error is present only in the Rate y axis measurement Errors are simple i e all data points have the same error Rate aN Substrate Double reciprocal Lineweaver Burk plot of the data above Note the large distortion of the error bars resulting from this plot 1 Rate 0 1 2 3 4 5 1 Substrate Here the error involved in the high data points of the transformed plot is much greater than in the low data points this is simply a consequence of the reciprocal transformation Linear regression unless suitable compensation is made for the distorted errors is therefore not an appropriate way to analyze the data and the original data should be fitted to the appropriate non linear equation Information Obtained from Data Fitting Data fitting provides the following information regarding the experimental data that are analyzed 1 The values of the parameters in the equation used that best describe the data These parameters may include for example rate constants inhibition constants etc whose values need to be determined 2 The accuracy of these calculated parameters From this information it is possible to judge how well the data fit the experimental model equation that was used In most cases however it is the numerical values of the parameters that are important and whose values are required from the analysis Multiple Regression 222 The most common form of non lin
13. Fonts Indents Y Chi change 1 Minimum iterations 2 Maximum iterations 20 The Fitting Criteria pane of the Non linear Fit item Store settings as defaults _ Restore GraFit defaults Fitting Criteria GraFit uses three separate criteria to decide when the calculations are acceptable Criterion Meaning Chi change This setting is optional and is activated by checking the Chi change box When selected fitting will terminate when the change in the 2 value is less than the percentage value entered providing the minimum number of iterations has been reached Minimum iterations GraFit will always perform the specified number of iterations 96 Chapter 6 Non linear Fit Item Criterion Meaning Maximum iterations Fitting will terminate when the specified number of iterations has been reached irrespective of whether the designated change in y2 has taken place The maximum iterations limit serves to guard against the possibility that the calculations will never reach a minimum and so forces control to return to the program eventually The settings can be stored as the defaults by clicking the Store settings as defaults button GraFit will remember these defaults next time the program starts Results Pane The Results pane holds a summary of the fitting results Ea Non linear Fit Settings Constants Estimatrix Results Number Format Fonts Indents X column Substrate X Y column Rate M 4 PU
14. Function ParamName index As Long As String Chapter 24 Passing Data Between Applications Description Get the chisq value for fit fit number Get the value for constant index Get the name for constant index Get the correlation coefficient for fit fit number Get the associated data table name Get the associated equation name Get the data format Get the name of column holding the error values Get the fitting status Get the weighting type Get the name of column holding the x data values Get the calculated y value for fit fit number x value Get the name of column holding the y data values Is this a linear fit Is robust weighting used Is there a limited x range Get the order of the linear fit Get the number of constants Get the number of data points for fit fit number Get the number of fits Get the number of parameters Get the value for parameter index fit Number Get the name for parameter index 319 GraFit User s Guide 320 GraFit FitResult Member Description Function RedChiSq fitNo As Long As Double Get the reduced chisq value for fit fit number Function StdError index As Long fitNo As Get the standard error value for Long As Variant parameter index fit Number Function XRangeLimitFrom As Double Get the lower limit of the x range Function XRangeLimitTo As Double Get the upper limit of the x range GraFit Helper Routines The file GF_module bas found in the GraFit Sam
15. GraFit User s Guide 2 Choose the Clear command or press DEL 2 a 4 5 E E O a Data in the selected region are cleared leaving a gap in the table Deleting a Region of Data Select the data value s or columns or rows to be cleared Choose the Delete command on the Table tab When deleting a range of cells the data values are moved up so that no gap appears in the table When deleting a column the remaining columns to the right of the selection are moved along to fill the gap Inserting Values into the Table 62 Values are inserted into the data table using one of the Insert commands on the Table tab Command Action Insert Cells Inserts blank cells in place of the current selection Cells are moved down to accommodate the new entries Insert Column Inserts blank columns in place of the current selection Columns are moved to the right to accommodate the new entries Insert Row Inserts blank rows in place of the current selection Rows are moved down to accommodate the new entries The Table Delete command in legacy mode The Table Insert commands in legacy mode Chapter 5 Data Tables Sorting Data The data in one or more columns can be sorted into ascending or descending order using the Sort command on the Table tab after first selecting the columns that you wish to sort Sort Data Index column Column s to sort Time 2 Amount 1 columnis selected Descending
16. UY 46 gt Robin My Documents GraFit Data v 3 Search GraFit Data Organize v New folder E G GraFit 7 0 Name Date modified Type Size IE GraFit Templates 34 Sample Data 21 02 2009 00 20 Shortcut LANI 48 GraFit Data Y Favorites 2 Recently Changed Li Public E F drive Backup Z E Computer MZ Desktop Documentation ERITHACUSNEW de Downloads Erith ERITHACUSNEW zE T File name Untitled Save as type GraFit Workspace Files er emplate Files GraFit Workspace File versions 5 6 a Hide Folders All Files l Chapter 4 Working with Files Loading and Saving Individual GraFit Items Saving Items The GraFit workspace comprises a number of individual items such as data tables graphs and analyses These individual items are saved in one of the following ways e Select the item in the Items pane or click on the item window and then select the Store Stock Item command Items are stored as files that have a GFC file extension By default items should be saved in the item store directory or a subdirectory off this The location of the item store directory is specified using the Options command on the View tab Loading Items Stored items can be loaded using the Add Stock Item command Creating a New File To reset the GraFit workspace e Select the File Close command Importing Data Files Data can be imported into GraFit to create a new data
17. Working A80 190 Data 1 Working A93 1103 Data 1 4 In many cases it will only be necessary to transfer a single region of data using the Data 1 part of the table If required however a second data region can be specified by completing the Data 2 entries should these not be required then leave the entries blank Results Section When the data are copied into the GraFit file GraFit will automatically carry out the data fitting procedures that are built into the file Once these calculations are complete the results can then be transferred back to Excel The Results section allows one to specify the name of the data fitting item the number of fitting results 1 or more and the number of parameters that are involved These are then copied back into Excel on the worksheet page that is entered at a location that starts at the cell specified Top Left E Fl Source aa Destination Excel F po Fit Full 8 4 Output A2 4 fe L Lpa P Specifying the Results locations for the Fit Full 8 4 Output A11 F Fit Full 8 4 Output A20 Fit first results region on the GF settings Fit Full 8 4 Output A29 a page Fit Full 8 4 Output A38 e Fit Full 8 4 Output A47 Fig Fit Full 8 4 Output A56 Fif Fit Full 8 4 Output A65 gt gt AO a a od re 4 The results are copied using a separate row for each parameter with columns containing alternating fitted parameter values and fitted standard error values for each
18. ccccsssccccsseceeeeeseceeeeseesees 224 Dara VCH A 225 Plotting Results from Multiple Regression cccccccssssscccccseseeceeseeseeeesseeeeees 226 TICO canta tool tio 227 NA E O doadhcuee pbessuamnadeomingeeeeeases 227 ASSUMPTIONS in Regression Analysis ooccccccoccnnonocnnncnnnnnnnononnnnnnnnnnnnnnnnnannnnnnnnnnnnnnos 228 Mear RESTO ta Ria 230 Wee UN a 231 Wele tine TYPE sii 231 NOn liN ar RESTESSION nui dicas 232 Standard Errors of the Calculated Parameters ccccoooccnncccnoncnnnncnannnnnononacnnnnnns 233 RODUSE WEIS ALING iio 233 Determining the Most Appropriate Weighting cccooccccccocnnncnoconononnnnnnanoncnnanonnnnnos 234 PIOT Of RESIGUA ERP OWS mural 235 Testine Goodness OF FIG TeSt oe alii 235 Nested Mode Sian Ii de 236 Comparison of Equations with Equal Numbers of Parameters cccceseeee 236 Mean and Standard Deviation ccccccssscccsseccccessccccenececsensecseecesseeeesseueeessueeeesaaees 236 Worked Data Fitting Examples sccsccsccsccsccsccsccsccsccsccsccsccscescescescescescesceccecceccsccnconees 237 Usne One OF ThE EQUATIONS Provided coil 237 PICCHU el 1 6 poe A o a bare asain danctate uaunesassoanieeseouecmuessoneee 238 CHECKING THEW GIB MEINE oia A beaten sos acetate 242 DraWine Residual Plots A aa 242 USING RODUSE Wee MUING daa dido 245 Detinine a New Equation lidad 246 10 Defining the Oxygen Binding Equation oooccccncccnncnnnnncnncnnnnnnnnccnn
19. kyt hot y Aoa 1 Ao 2 E l Here the two processes have separate A and k values T Double Exponential phase 1 This displays the first of the two phases in isolation T Double Exponential phase 2 This displays the second of the two phases in isolation 2 Double Exponential with offset This equation is used when a double exponential process decays to a non zero limit value 2 Single Exponential s Bal The equation for a single exponential J decay is B al Double Decay 3 47 y Ao emt i The observed values decay with time t from an initial value A at t 0 to O at t 0 1 2 3 4 5 The rate constant for the decay is k The half life for the process T is given by log 2 k To display a linearized plot of this equation set the y axis to have a logarithmic scale Single Exponential with offset If the observed values do not decay to 0 but to some defined offset or background value the equation for a single exponential process is y Ap e 1 offset In this equation offset is the limit of the decay 2 Triple Exponential For a triple exponential decay process the equation is y Aoa eit Aga e Kat Aga est Chapter 22 Equations Provided Triple Exponential with offset This equation is used when a triple exponential process decays to a non zero limit value IC 50 These equations are used for analyzing radio immuno
20. AY Graph Properties Genera it une has Seat Y ads selector Tick settings a gt Sze Axis offset Distance fom em frame Tick marks Width 0 8 pt Coir MOM Style Width 0 8 pt Color Ld Minor grid Width 0 8 pt Color L _ F Chapter 15 Using Multiple Graph Axes The Ticks page also includes an Axis selector section which allows each different axis to be selected After choosing the axis to modify alter the tick settings as required 197 GraFit User s Guide 198 CHAPTER 16 Column and Bar Charts The Chart Wizard Chapter 12 described the basics of creating graphs Selection of Column Chart or Bar Chart produces the Chart Wizard to assist with the setup all settings can be adjusted later if required Column and bar charts differ only in their orientation and so the same process is carried out for each The process of editing charts is broadly similar to editing X Y graphs and Chapter 14 should be consulted for further information Select Data Source Page Chart Wizard Select Data Source Legend text Data set 4 Source Category axis Upper data Error column E Lower data The Select Data Source page allows you to choose the source and type of data to plot 199 GraFit User s Guide 200 Legend text The data set plotted is associated with an entry in the data legend The entry shows the symbol used to represent the data and a
21. Absolute error bars on the x axis proportional errors on y Y Error bars This section is used to specify the type of errors present on the y axis The settings are analogous to those described above for x error bars Axis Association Page Fitted Data Properties 1 Scale Oto 10 The Axis Association page allows the data to be associated with the various axes that are present see Chapter 15 for information on multiple axes XY graphs can contain many 183 GraFit User s Guide different x and y axes and the data can be associated with one the minimum or many different axis scales Check the boxes of all axes that you wish to be associated with this data set Equation Page equation data only Curve Properties This page selects the equation that describes the curve to be plotted All equation items present in the GraFit workspace are listed 184 Parameters Page equation data only Curve Properties Equation Constants Axis Association Parameters gt Read from table Column Direct entry Chapter 14 X Y Scatter Graphs This page is equivalent to the Curve Parameters page of the XY Graph Wizard see page 162 for further information Constants Page equation data only Curve Properties Column 185 GraFit User s Guide This page is equivalent to the Curve Constants page of the XY Graph Wizard see page 162 for further information This page is present even for equa
22. Equation Item test 2 contains a minus sign ten contains a punctuation character e No distinction is made between upper and lower case characters within a symbol definition Therefore the symbol names grafit GraFit GRAFIT and gRAfIT are all considered identical We recommend using meaningful names wherever possible when defining symbol names Using Final to represent the final concentration of a substance rather than z will make understanding your definitions far easier for others as well as yourself In addition it is often useful to add a full descriptive name Number of X Variables After entering an x variable definition the data entry grid will show a blank entry position immediately below the defined variable If you enter a second definition in this location a further blank entry position will appear The number of rows of information that are entered into the grid therefore defines the number of x variables in the equation When to use Multiple X Variables Most equations are of the form y f x and so have just a single independent x variable In equations with multiple independent variables the observed value y varies as a function of more than one variable condition For example the rate of an enzyme reaction may vary as a function of both substrate and inhibitor concentrations In this case there are two independent x variables In principle it is possible to have any number of independent variables although
23. Graphs 139 Adding data 186 Aligning objects 145 Arrow properties 152 Axis association 183 Axis scale 172 Axis text 174 Chart bar style 203 Colors 146 Creating 133 Curve resolution 163 Data point style 204 Data point styles 161 Data curve properties 176 Default text properties 147 Duplicating objects 143 Error plots 159 181 200 208 Exporting 154 Fill properties 150 Index Font properties 153 Grid settings 169 Grouping objects 145 Histogram bar style 210 Initial scaling 208 Initial scaling 159 Join style 205 Join styles 164 Legend 157 200 207 Legend box 174 186 Line properties 151 Location 172 Main graph region 166 Multiple axes 189 Object locations 152 Plotting equations 162 Rotation properties 151 Scaling 201 Selectable regions 166 Selecting objects 142 Sizing objects 143 Split axes 189 Text 147 Text objects 143 Tick styles 160 202 209 Transformations 159 Visibility 167 X scale 170 Y scale 172 Y ticks 171 Z ordering 146 Grid Lines command 140 Grid Magnification 65 Group command 145 H Hard coded Equation definition 325 Hard coded Equations Enabling 325 Inserting 326 Library Files 327 Hardware Requirements 17 337 GraFit User s Guide Headers 42 Help commands 42 Help menu Contents command 43 Henderson Hasselbalch equation 237 Hide Object command 167 194 Hill Plot 270 Histogram 136 207 Histogram W
24. The Welcome screen will load automatically Note Some systems are configured so that automatic running of CD ROM software is disabled If your system is set up in this way the Welcome screen can be displayed by selecting the Run command from the Windows Start menu and entering the command D CDRUN where D is the drive letter of your CD ROM 17 GraFit User s Guide Installation Process GraFit Select the Install GraFit option to install GraFit onto your computer The installation program will copy the files to your hard disk and place an entry for the GraFit program on the Windows Start menu under the Programs entry Adobe Acrobat Reader The GraFit installation includes this user manual saved in Adobe Portable Document Format PDF To read this file it is necessary to have the Adobe Acrobat Reader version 6 or later installed If you do not already have Acrobat installed you can do so by selecting Install Acrobat Reader Installation of Acrobat Reader requires your computer to be connected to Internet Activating the GraFit Program After installing GraFit it is possible to run the software in evaluation mode for 14 days During this time the program is fully functional and is equivalent to a single user activation To continue running the program after the evaluation period expires it is necessary to activate the program online by entering a valid activation code serial number Activation codes are purchased after the
25. clicking the Forgotten password button will automatically email this to you Multi user Installations If you have a multi user installation then there is no Forgotten password button as passwords are administered by the designated License Administrator see later The License Administrator is also able to deactivate any multi user installation remotely Changing Computers If you change computer it is advisable to deactivate your GraFit installation from the old computer before reinstalling the program on the new computer Checking for Software Updates You can check to see whether any program updates are available by running the Help gt Check for Updates command If updates are available online you have the opportunity of downloading and installing them Uninstalling GraFit GraFit can be removed from your system by the following procedure 1 From the Windows Start menu launch the Control Panel application 2 Double click the Add or Remove Programs icon 3 Choose GraFit 7 to uninstall and click the Change Remove button 4 Follow the instructions Note We strongly recommend that you deactivate GraFit as described above prior to uninstalling the program 21 GraFit User s Guide Different GraFit Editions 22 GraFit comes in a variety of editions that share key functionality but have different licensing and activation models The different editions are listed below Edition Features License Validity GraFit Pro
26. described below then selecting the Calculate gt Fit Data command on the Home tab After fitting the data the results can viewed from the Results pane Status Bar Indicators When a fitting item is selected the status bar shows the current fitting status indicating whether the item is currently Fitted or Not Fitted In addition the status bar shows when a calculation is in progress Whenever any calculation is being processed the calculation pane shows an animated abacus Fitting status pane Calculation pane t The Calculate gt Fit Data command in legacy mode 103 GraFit User s Guide Settings Pane 104 The Settings pane is used to specify the main details of the fit Linear Fit Settings Results Number Format Fonts Indents Equation y a bx Linear Regression Weighting Simple Data table Data The Settings pane of Data layout x Ly the Linear Fit item X data column x Data ho E Limit x range data column y Data T Equation Choose the equation to use for the data analysis All the available equations are linear and are of the form given below y a bx cx dx The default linear regression equation is y a bx Weighting All types of regression analysis require information about the error distribution in your data The reason for this is explained fully in Chapter 20 but briefly it is necessary to specify whether the inaccuracies are like
27. sqrt x The square root of x Vx tan x Tangent of x The expression within the brackets may be of arbitrary complexity for example Seam k sgart 1 All the geometric functions sin cos etc use radians as their units The following predefined constants are also available Constant Value _pi_ T 3 1415926536 e e 2 7182818285 _rnd_ A random number between 0 0 and 1 0 Messi A random number with Gaussian error distribution The number has a zero mean and unit variance Multi line Equation Definitions In its simplest form as described earlier the equation definition is a single line that calculates the y value For example if the equation to be used was y ax b then the equation definition might be written as Here a and b have been declared to be the symbols that represent the parameters x has been declared to be the symbol that represents the x data and y the symbol that represents the y data Note that the multiplication between a and x needs the explicit inclusion of the multiplication operator The equation definition can also be defined over several lines in order to simplify a complicated definition This allows the calculation to be broken up into several steps Use of Temporary Variables Temporary variables can be used to hold intermediate calculation results and simplify definitions For example the following definition uses three temporary variables a b and c 119 GraFit User s Guide
28. 0000 1 5000 2 0000 2 5000 3 0000 3 5000 Clicking the mouse within the row selection area results in the entire row being selected To make a more extensive selection e Click on the start position e Use the scroll bars to move the window as necessary e Hold down the SHIFT key then click on the end position for the selection Discontinuous selection e Hold down the CTRL key and click the required entries 58 Chapter 5 Data Tables Selecting Entire Columns Whole columns of data are selected by clicking on the column selection region The selected column is displayed inverted Substrate 0 2500 1 0000 0 5000 1 5000 1 0000 2 0000 1 5000 3 7000 2 0000 4 1000 2 5000 4 5000 3 0000 5 0000 3 5000 5 5000 md Column selection region 2 3 4 5 E E O 4 Drag the mouse along the column selection region to extend the selection In a similar fashion clicking in the row selection region allows whole rows to be selected Copying and Pasting Data Selected regions of the data table are copied to the Clipboard using the Copy command and pasted using Paste Data may also be pasted to and from other applications in the same Way Copying and Pasting Regions of the Data Table 1 Select the region of data that you wish to copy 2 Select the Copy command 3 Move the data entry position to where the data is to be copied 4 Choose Paste The data will be inserted at the data entry position Copying
29. 120 Eo pal E ao eo ity oO oo A pa es a The remaining symbols Kd total Capacity are defined in the equation editor as parameters or constants Values are assigned to the temporary variable using the assignment operator after which they may be used in expressions as normal Use of temporary variables makes the above definition more easily read and speeds up the calculation as the value for b need only be calculated once Each line of the definition is separated from the next by pressing the RETURN key Conditional Statements The values that are evaluated can be controlled using the If statement This takes the form tecno nea statements Ie Conc canoa Wasa statements For example it is possible to return different values to the program depending upon the value of a parameter constant or independent variable If extra 1 Then Return al To px lehiqve alae If extra 2 Then RS E Sa Ore ee Endir If extra 3 Then Retinal a Nr os EMALE FRercurn ON Comments Comments can be added to annotate the equation definitions Anything after an apostrophe is a comment pas Ms anyrning atter a semi colon Chapter 9 Equation Item Looping Statements The For statement has the following syntax For varname startValue To endValue statements For varname startValue To endValue Step stepValue SES Seis Goto Statement Another statement that will be familiar to programmers is the Goto stateme
30. Data Transfer TYPE ds 213 Embedding and Linking without using the Clipboard cooooccccnoccnnnnonnnnnns 214 Embedding and Linking with GraFit ooocccccoccnncnonnnnnnnnnnnnnnnnononnnnnnnnnnnnnanonnnnanonnnnnos 214 OraFitas aN OLE CONTAINGK ias 214 Graritas an OLE SOUCO iaa 215 GraFit User s Guide Data Fitting BASICS oaair ikea caccussatassusacsessvncasecslwaccncshosasanebenspausbovapeesbesesausbnsesnnateatians 217 When Is Data Fitting Appropriate orcima iniaa a 217 A A 217 NESESITO decis 219 Practical CONSI ALON eusann 219 Types of ResressiomAnalVS Sida aan 220 LIN Gal Reese 220 Polynomial Regression ccccsseccccsseccccsscecseececcesccssescesauesecseuecesseaseessaeeeeseas 220 N n inear estes SiN la 220 Additional Considerations for Non linear RegressiON oocccccccnocnnnncconcnnnnnnnnnos 220 WEEIB DUNE Sche ME asa RES 221 Linear Regression of Transformed Data a dd 221 Information Obtained from Data Fitting ccccooocccnnccnonnnnnnonanonnnnnnnnonnnnnnnocnnnnnnnnos 222 Multiple Regreso di 222 Uses for Multiple Regression oocccccccoccnnnnconoconnnonononnnnnnnncnnnnnonnnonnnononcnnnnnnnnoos 223 Advantages of Multiple Regression oocccccccnocccnnconononnnnnnnccnnnnnnanononnnnnnnnnnnnnnnos 224 Disadvantages of Multiple Regression cccccssssscccccessecceeceeecceesaaeecessaeneees 224 Defining EQUATIONS iia didas 224 Fitting Data by Multiple Non linear Regression
31. Format Fonts Indents Settings Constants Estimatrix Equation Enzyme Kinetics Y Weighting Simple Robust aa The Settings pane of Data layout E D the Non linear Fit item X data column Substrate Substrate Limit x range Y data column tv Equation Choose the equation to use for the data analysis The equation must exist as a separate item within the main GraFit window See Chapter 9 for information about equation items Weighting All types of regression analysis require information about the error distribution in your data The reason for this is explained fully in Chapter 20 but briefly it is necessary to specify whether the inaccuracies are likely to be the same for each data point Simple weighting are proportional to the value of the y data points Proportional weighting or have been individually estimated Explicit weighting The default Simple weighting is often the most appropriate setting Robust weighting is additional weighting that provides a way of removing outliers see Chapter 20 Check the Robust box to add robust weighting to the fit Data Table The data to be fitted must reside in a separate data table item within the GraFit workspace Select the table that holds the data from this drop down list Data Layout The data layout specifies how the data are arranged for data fitting The default layout as shown in the figure above is that the x and y data c
32. MR eo Lo GraFit Pro Academic cokane MA 12 28 2009 Do Qerithacus com 1 10 r rd Ah e PI Help A A A A A A a a O ee List of all software licenses administered via an individual License Control Panel account Managing a Multi user License To manage an individual multi user license clicking on the entry in the list above This will produce a screen that shows information about each activated user Software details for Activation Code MAW 000 Software From Company Activation Date Activation Email License Type Expiry Date GraFit Pro Academic Erithacus Software 12 28 2009 O erithacus com pra j 12 28 2010 Resend Password ba ta 4446 8 te Computer ID Seat Activation Date De activate Computer ID Seat Activation Date De activate ROBIN ADVENT 12 28 2009 Yes S i Pp O A E Management of an individual multi user license 27 GraFit User s Guide All activated seats are listed by Computer ID which is the name of the individual computer that has activated the software Changing the Activation Password and Administrator Email Address To change the Administrator Email address click the Change Email button and to update the password click Change Password If you change the password then all new activations will need the updated password Changing the password requires knowledge of the current password if you unsure of this click Resend Password to email this to the current Administrator Email address Deleting a User fro
33. Multi user version with web based Rolling 1 year management of activation codes subscription extendable GraFit Pro As above for use in non profit organizations Rolling 1 year Academic only subscription extendable GraFit Single user stand alone version of GraFit Unlimited GraFit As above for use in non profit organizations Unlimited Academic only GraFit Student Entry level single user student edition for 1 year subscription use in non profit organizations only single user license You can tell which edition of GraFit you have from the appearance of the program title bar Edition Appearance GraFit Pro f ko gt GraFit Pro Untitled GraFit Pro Academic GraFit GraFit Academic GraFit Student Academic Licenses If you work in a not for profit organization then you can purchase an Academic GraFit license at a reduced price Academic licenses are restricted in that they cannot be used in a commercial organization they are available for both single user and multi user installations Student Licenses The GraFit Student edition is a low cost entry version that is specifically designed to support use by students This edition is restricted to use only in not for profit organizations The Student edition has a number of in built licensing restrictions it can be activated only on a single computer at any one time and the license expires after 1 year Continued use beyond that time will require purchase of a new
34. Right To Left ta 4d Animation v Frame Theme Changing from ribbon to menu and Changing from menu and toolbars to ribbon toolbars 37 GraFit User s Guide The GraFit Workspace When initially opened the GraFit workspace is empty A GraFit file can comprise one or more data tables graphs data analyses equation definitions transformation definitions notepads and embedded objects Collectively these items make up the GraFit workspace which are stored to disk as a single file After adding a number of items to the workspace the appearance of the GraFit window is shown below Items pane Active item Item window selection window tabs G4 Select All fal Edit Text Undo Redo Cut Copy Clear All Clipboard Edit ax pat Fonts h Indents Estimatrix Constants Definition Estimates Indents General X Variables arameters X A GraFit workspace with a number of items added In this example one each of the following items has been added a data table a graph window a non linear fitting item a linear fitting item an equation definition The linear fit and graph items have been closed so that their windows are not visible in the workspace their icons within the Items pane are drawn lighter to reflect this All the added items are stored inside the same GraFit workspace and can be saved and loaded from disk using the File menu commands If you run a second cop
35. Roy Soc London Ser B 144 29 54 Chapter 21 Worked Data Fitting Examples Defining the Oxygen Binding Equation To define this equation we need to create a new definition in our workspace using the Add Equation command See Chapter 9 for a complete description of defining equations Definition of the new equation requires that we make appropriate entries in this item window X Variables Symbol p p Parameters Symbol K K1 K K2 K K3 K K4 Y name Sat Definition Sat Description po2 Description Kl value K2 value K3 value K4 value 100 K1 p 2 K1 K2 p p 3 K1 K2 K3 p p p 4 K1 K2 K3 K4 p p p p eC ce a ap Re oo A RILAR AO Op dE Kil Kh2 Kho Kh4 p po DABI The appearance of the equation definition item after entering this definition and changing the name of the item is shown below 95 As this definition is for demonstration purposes only it is defined in this way as this is the nearest form to that of the equation given Although the definition would function in this form it is rather inefficient to write it like this and a better approach would be to enter the mathematically simpler Sat 1 00 Kio 1 Y K2 p 2 Ba pot Kapa 7 C4250 Kl p 1 K2 p 1 K3 p 1 K4 p 247 GraFit User s Guide Oxygen binding Le s Constants H Estimates Fonts Indents Sat 100 K1 p 2 K1 K2 p p 3 K1 K2 E3 p p 4 Symbols double dick to insert into
36. The fill style applies to the graph background Line Page The line style applies to the frame drawn around the graph as set on the General page It does not apply to the color of the tick marks which are set on the appropriate tick page 68 The Edit Hide Object command in legacy mode 167 GraFit User s Guide X Ticks Page AY Graph Properties X ads selector Tick marks X ads H 1 Width 0 8 pt gt Tick settings _ o uno Sie Otome Size Medium Style 2 No line Axis offset Distance from frame X axis selector X Y graphs can have multiple x axes and the tick settings for each are set individually Set the x axis number from this box when first created only a single x axis will be available Tick settings Ticks can be drawn on the inside outside both sides or neither side of the Upper graph edge the Lower graph edge or on the X axis itself assuming the scale settings are such that this lies in the center of the graph The tick sizes can be set to small medium or large which are scaled relative to the overall graph width or to specific where the exact tick size is entered into the edit field li Left Lower ticks outside Left Lower ticks inside Left Lower ticks both sides 168 Chapter 14 X Y Scatter Graphs Axis offset The axis offset controls the distance that the axis scale is drawn from the frame If the offset is O the scale is drawn immediately adjacent to the central
37. This is the first activation of a multi seat activation code The email address you enter will be identified as the Administrator for this activation code Activation password U Please enter and confirm a password that you wish to associate with your activation In the event that you need to reactivate your software you will need your password as well as your activation key Activation Information dialog box for multi user activation codes first activation Password Confirm password Activation email address U Please enter and confirm your email address This is required so that your password can be emailed to you if you forget it Email Confirm email The activation password that is entered will be used to activate all further seats associated with this license code and the email address entered will be that of the License Administrator who will be able to monitor license use via the License Control Panel LCP see below Note Activation of the first copy of GraFit will use up one of the available license seats If the License Administrator is not a regular GraFit user for example in a large corporation this could be an IT professional then it is possible to regain use of the seat by deactivating this user installation via the License Control Panel Deactivating an individual user in the License Control Panel will not remove their registration as the License Administrator Setting up the License Control Panel A
38. a matrix of permutations is tested It is important to limit this grid search to a sensible number of combinations for example if 10 parameters were present and 10 steps were selected for each this would involve 10 values to be tested clearly an unrealistic number When data fitting starts each permutation is tested and the combination that gives the best fit lowest Y value is used as the starting estimates for non linear regression If both automatic and manual estimates are present each is tested and the best of the two is used as the starting estimates for regression Chapter 6 Non linear Fit Item Linking Values to Data Table Entries It is possible to obtain the estimate values from entries within a data table Data table entries can be updated as a result of calculations and can even further linked to values in external data files To link values in this way click the Link to table checkbox and select the data table that holds the data plus the columns that are the source of the data values Interactive Estimator Finding suitable initial estimates can be a laborious task and to simplify things GraFit provides an interactive visual tool that is accessed by pressing the Interactive Estimator button The Interactive Estimator dialog is shown below Interactive Estimator Graph scale Amin Xman 9 Ymin 2 7 The Interactive Ymac 63 i Estimator dialog Substrate TERE Estimated value A simple graph of the d
39. a multi user license can be re allocated to other users by deactivating one or more of the installed seats Deactivation can either be performed by the License 28 Chapter 2 Installation and Activation Administrator from the LCP or by the using the Deactivate button that is found on the Help About dialog box Note Deactivation of a seat will typically take 24 hours to process You will therefore not be able to add a new user seat until this time has elapsed Expiry of Multi user Licenses Multi user licenses are sold on a time limited basis and expire one year after the initial activation Extension of the license needs to be performed before the expiry date in order to ensure all users are able to continue using the software When does a License Expire The length of time remaining on the license can be found on the Help About dialog box Serial noz MU Matte The expiry of a multi user license is shown on the Help About dialog box Your multi user software license expires in 363 days time Three weeks before your multi user license is due to expire all users of the license will see a warning banner whenever GraFit is run GraFit Pro Untitled Home Text View Options Ey 7 ata Lj Import Data y oar Ca Select All a L Add Stock Item dd a A Gell Edit Text d a 7 ley Paste Undo Redo Cut Copy Clear Object r way 2tore stock Item r Items Clipboard Edit Object T Multi user license
40. absorbance may not be zero the E V equation becomes i 0 DE At Aw 1 e offset A REA T 1st order offset decay curve This transformation plots the rate equation as an exponential decay Use it with Decay Curve Offset transformed data T 1st order offset show limit This transformation displays the limiting value for the equation containing an offset Chapter 22 Equations Provided T 1st order offset show offset Use this transformation to show the offset value 2 4st order rate versus S s To find enzyme kinetics parameters it is often necessary to carry out a series of first order rate reactions at different substrate concentrations The rate constant versus S is then analyzed using the Michaelis Menten equation Using this 3D equation allows these procedures Ting to be combined giving A Aw exp a 2 4st order rate versus S offset Amount This equation is the same as that above but also includes an unknown background value A Aw exp background 283 GraFit User s Guide 284 CHAPTER 23 Standard Templates Data fitting templates provide a quick way of analyzing data and plotting graphs In general you are recommended to construct your own specific templates that are best suited to the data you wish to analyze However GraFit comes with a few standard templates that can be used for common data fitting si
41. access the internet using a browser such as Microsoft Internet Explorer GraFit will automatically make use of any proxy settings that might be needed In some rare circumstances it may be necessary to use custom proxy settings in order to access the internet these can be set by the Change Proxy Settings button Running in Evaluation Mode The software can be evaluated for 14 days prior to purchase To start your evaluation period click the Evaluate button on the Software Activation dialog box In order to start the evaluation your computer must be able to access the internet The first time you evaluate the software you will need to supply information about where the Evaluation code should be delivered This code will be emailed to you at the address entered into the following dialog box Get Evaluation Activation Code Evaluation email address U Please enter and confirm the email address that your evaluation activation code will be sent to When you first start the evaluation period you must enter the email address to which the evaluation activation code will be sent Click OK to request an evaluation activation code This code will be sent immediately but you should allow a few minutes for your email system to process the message Note Most email systems are set up to move unsolicited email to a Junk email folder In the event that you cannot find your evaluation code email try looking in your Junk folder in case it has been mov
42. allowed It is also possible as shown above to include an OLE object embedded in the text In this particular example a Microsoft Equation object has been embedded within the text Note The Microsoft Equation editor which comes with Microsoft Word is not provided with GraFit Changing Font Styles To alter the style of a selected region of text use the commands available on the Font tab Inserting OLE Objects To insert an OLE object into the description text position the cursor where you wish the object to be placed and select the Object Insert New Object command from the Home tab The Edit Font command in legacy mode The Edit gt Insert New Object command in legacy mode Chapter 9 Equation Item Insert Object Create New Microsoft Equation 3 0 A Microsoft Graph Chart Create from File Microsoft Office Excel 97 2003 Work i caa Microsoft Office Excel Binary Worksh Microsoft Office Excel Chart g Microsoft Office Excel Macro Enablec Microsoft Office Excel Worksheet E Display As Icon Microsoft Office PowerPoint 97 2003 7 Inserts a new Microsoft Equation 3 0 object into a Choose the object type to create and then click OK Editing OLE Objects An inserted OLE object can be edited by double clicking on the object of by choosing the appropriate menu command from the Object menu on the Home tab X Variables Pane The X Variables pane is used to define the names of the i
43. also available by right clicking on the object Comparison of Data Transfer Types The following table compares the various ways that data can be transferred between applications via the Clipboard How to paste information in this format Advantages Disadvantages Non OLE Clipboard transfer For non OLE applications Edit Paste For OLE applications Edit Paste Special and select a non OLE format Compact data format No editing allowed after pasting Embedding For OLE applications Edit Paste Allows information to be edited after pasting Pasted information is stored within the target document No link maintained with the source document Documents containing embedded objects tend to be large Linking For OLE applications Edit Paste Link or Edit Paste Special and choose to paste as a link Allows information to be edited after pasting Since data are kept within the source file this allows efficient use of space when several documents may wish to link to the same object Linked information is kept in a separate file which may become lost Note The default action for a Windows application that supports OLE is to embed information whenever possible if the Edit Paste command is used As described in the table above embedding is not always the most appropriate way of sharing the information and linking is frequently more desirable for which you need to sele
44. are displayed set Trailing zeros in fraction to Show 0 s Enter the maximum number of digits to use when displaying numbers Check this box to include a grouping separator The symbol used for this separator and the number of digits in the group are set for the Options command on the View tab Check this box to display the values using scientific format e g 2 435e003 instead of 2435 000 Check this box to ensure that small values which would otherwise be displayed as 0 0000 are shown in scientific format e g 2 0987e 008 Chapter 5 Data Tables Specification Meaning Trailing zeros in Chooses the way that trailing zeros are displayed in the formatted fraction numbers It is possible to show the 0 characters to omit them orto replace them with spaces Save As Defaults Stores the current settings as the default for future GraFit sessions Restore Defaults Restores the current GraFit formatting defaults to the selected columns Magnification The magnification of the data table text can be adjusted to allow more or less values to be visible Magnification is controlled by the Zoom command found on the View tab Changing the magnification and altering the font size Preferences command on the Table tab both modify the size of text displayed on screen However changing font size using Preferences also changes the size of printed text while Zoom only affects the on screen appearance Using the
45. be used In addition it is possible to use an icon image that resides in any executable or DLL file on your computer Item specific Commands Although some commands apply to all GraFit items many are item specific For example the Fit Data command applies to the Linear fit and Non linear fit items but not to the Data table or Graph items Commands that are not available are shown grayed In addition each item window has one or more specific menus that are only present when this window is selected For example only graph items have Graph and Object menus Printing the Item Contents 40 The contents of any item window can be printed using the File Print command and the printing previewed using the File Print Preview command Found on the Window menu in legacy mode G Eee a Le ei A Home Text View Table Pas Te As 5 Import Data 7 x G4 CI G ada Stock Item ai Go 2 A 4 Undo gt E Store Stock Item Items e Print gt 4 GO A A Y Close Preview Items ax E Data x E E Data 2 2 0000 3 3 0000 4 4 0000 5 5 0000 6 6 0000 in 7 0000 8 8 0000 9 9 0000 10 10 0000 11 Ready Page Settings Object Y Edit Object 10 0000 Chapter 3 GraFit Basics Print preview The File Print gt Page Setup command allows the paper size and orientation to be specified and the margin sizes to be entered These settings are stored separately for each ite
46. command the Text Field dialog box will be populated with the currently selected field code settings General Object Properties Selecting the Properties command on the Graph tab or double clicking on a graph object shows the object properties dialog box This dialog box contains a number of pages some which are common to many objects and some which are specific to the object selected The common property pages are shown below specific property pages discussed later Fill Properties Rectangle Properties 150 Chapter 13 Graph Item Check the Fill box to fill the object with the selected color If this box is unchecked the object will be transparent Line Properties Rectangle Properties The Line page sets the width style and color of the line It is possible to set the Style to No line if you do not wish a line to be drawn Rotation Properties Rectangle Properties Rotation Degrees Curent rotation is 0 0 degrees 151 GraFit User s Guide Objects can be rotated and their rotation is specified from the Rotation page Rotation is entered in degrees which is measured in a counter clockwise direction The rotation can be set as an absolute figure or relative to the current rotation Location Properties Rectangle Properties Relative to Size 3 334 cm 2 988 cm Maintain aspect ratio The location of the selected object is set from the Location page This allows the object position t
47. constant The i Jandu S K Ray S R Brooks L amp Leatherbarrow R J 1990 Biochemistry 29 6265 6269 259 GraFit User s Guide dataset column x must hold the value 1 0 if the x and y columns are from the first data set and 2 0 if they are from the second Clearly this can be extended to further data sets if required The value a in the above definition is a temporary value used to simplify the calculation The figure below shows the guanidinium hydrochloride unfolding of a native protein and of a mutant variant of this protein The mutation has caused the protein to become less stable making it unfold at lower concentrations of denaturant The solid lines indicate the fitted curves using the above equation The difference in stability can therefore be determined 1 2 O Native 1 0 a 8 E e Mutant gt 08 0 p Q 06 a O o 0 4 l LL 0 2 p e 0 1 2 3 4 5 6 GdnHCI Enzyme Inhibition Samples 260 The final set of samples concern the analysis of enzyme inhibition data These sample files are relatively complex and provide the basis for your own enzyme inhibition analyses If you need to fit inhibition data we recommend that you examine the files to see how they analyses have been set up Competitive Inhibition Fitting The files Competitive Inhibition and Competitive Inhibition LB show how it is possible to fit data to a competitive inhibit
48. data entry position within the data table To Press Move the entry point to the right TAB Or RIGHT Move the entry point to the left SHIFT TAB Or LEFT Move the entry point up UP Move the entry point down DOWN Or RETURN Move to the first column of a row CTRL LEFT Move to the last column that contains data entries CTRL RIGHT Move to the first row of a column CTRL UP Move to the last row that contains data entries CTRL DOWN It is often convenient to press RETURN or DOWN between entering data points Note If you have a region of the table selected pressing RETURN will move the data entry position left to right within the selected region If you prefer to enter fx y data pairs in sequence X Yy Xz Y2 then it is convenient first to select the columns see Selecting Entire Columns that will hold the data and then to enter the values pressing RETURN between each data entry Moving the data entry position using the mouse To move the data entry position using the mouse move the mouse pointer to the position you require and click the left mouse button 57 GraFit User s Guide Selecting Regions of the Data Table Selecting Part of the Data Table To select a portion of the data table drag the mouse over the required region Alternatively hold down the SHIFT key and use the cursor keys to extend the selection The selected region is displayed shaded as shown below Substrate Selected region 0 2500 0 5000 1
49. definition X variables Parameters To fit these data add a new non linear fitting item using the Add gt Non linear fit command then complete the Settings page as below a Non linear Fit Estimatrix Equation Weighting Explicit user defined Robust Data table Data Data layout x y e X data column pO2 F Limit x range Y data column Sat Error column Note the use of Explicit weighting which requires the selection of the column that holds the error values The results of the analysis are as follows 248 Chapter 21 Worked Data Fitting Examples 100 80 c O p II g 60 Parameter Value Std Error D ee a epee Q 40 K 1 value 0 0962 0 0047 K 2 value 0 1017 0 0290 K 3 value 0 3467 0 1071 20 K 4 value 0 9176 0 0634 0 0 10 20 30 40 50 pO2 For comparison the results quoted by Roughton et al are given below Parameter Standard value Error K 0 0961 0 0049 K 0 1040 0 0293 K 0 3352 0 1018 K 0 9263 0 0648 It should be borne in mind that Roughton et al performed their regression analysis without the aid of personal computers or even pocket calculators Defining a New Equation 2 The hybridization of RNA and cDNA molecules is used to provide information about the genome organization and complexity of an organism This hybridization can be described by the equation n a ri gt P f e e 70 595 Rot Roto s p Do l i 1 Here d Do is the fractional hybridization at an
50. described later 229 GraFit User s Guide Linear Regression The simplest case for regression analysis is when the equation is linear For the linear equation y a bx y is defined as hey x gt So a bx In this equation ao is the standard deviation of the data point The best fit to this equation will yield parameter values for a and b that produce the minimum possible value of y This is equivalent to saying that the first derivative of y with respect to a and b is equal to zero 09 y 1 bx da a 0 AEA 1 2 ype 0 Oi 0 y 1 bx ab ab L No2 Vi AT 2 So a bx 0 If we rearrange these equations it is possible to solve for a and b exactly to give values for these parameters that minimize 2 This is shown in the equations below 52 y 1 0 a a l A yA Xi Vi Xi 2 2 Oi 0 Da Lee 1 0 0 j DA XiYi a a l l 230 Chapter 20 Theory L Xi y xiy a a l l y 1 Xi 0 a A Weighting This analysis presupposes that we know the standard deviation de for each data point In general we will not have such information available However only relative uncertainties need be known and if we assume that all the data points have the same inherent errors the equations simplify to give Ya De i Y muy be N Y y 1 an x Y x Y y E A gt Xi gt Xi Vi This assumption that the errors are constant is often made for exa
51. drawn at the end of the axis This setting is used when drawing split axis scales see Chapter 15 for more information about multiple axes Y Ticks Page The Y Ticks page controls the tick settings on the y axis in the same way as the X Ticks page controls the tick settings on the x axis see page 168 171 GraFit User s Guide Y Scale Page The Y Scale page controls the scale settings for the y axis in the same ways as the X Scale page controls scale settings on the x axis see page 170 Location Page This sets the location of the graph within the graph page Axis Scale Regions The axis scale region controls the display and formatting of the scale numbers Scale Page Axis Scale Properties Scale Font Location Number format Text Decimal places Alignment Right ka ax digis x Orientation bl Thousands separator E Scientific format E Format string E Power format Trailing zeros in fraction Values Omit O s Scaling power O Number format These settings determine the way that the numbers are displayed Setting Description Decimal places Enter the number of decimal places to display Note that trailing zeros will be removed if the Trailing zeros in fraction setting is Omit 0 s Max digits Enter the maximum number of digits to be used The displayed values will be rounded to ensure that only the requested precision is displayed 172 Chapter 14 X Y Scatter Graphs
52. equations provided These equations may be edited to see how they have been defined and further equations can be added This chapter briefly describes the equations that come with the program It does not attempt to discuss the detailed background of each equation as it is assumed that the user of a specific equation will be familiar with this The equations and transformations that come with the program are installed into the Group Item Store directory and can be accessed using the Add Stock Item command Equations are grouped into separate sub directories which appear as different pages on the Add Item dialog box Add Item Enzyme Inhibition Enzyme Kinetics ligand Bindng pH Rate 1C50 0 100 Ic50 IC50 IC50 show IC50 backgrou backgrou backgrou IC50 full 4 IC50 full 4 IC50 full 4 IC50 full 4 param Param sh param sh param sh 101 The File Add Stock Item command in legacy mode 265 GraFit User s Guide Equation and transformation definitions are distinguished by the nature of their icons The following sections describe the contents of these various pages 2 Substrates The definitions on this page are designed to analyze enzyme kinetic data involving two substrates Ping pong Use this equation where a ping pong mechanism applies The equation relating the rate of reaction v to the concentration of substrates A and B and the constants K and K is Vax lA B
53. file Alignment Number fe EMBED GraFit Graph 4 D E Enzy Clipboard S GraFit 1 v Enter Name for Selected GraFit Graph Naming an embedding GraFit item within the Excel file Embedded GraFit Graph SNOW O ha my Mr PA g ys ga T ef It is possible to embed multiple GraFit analyses within a single Excel file if required Chapter 24 Passing Data Between Applications Note During data fitting when passing data to an embedded GraFit file there is a short delay as each file is opened This means it can be inefficient to use very large numbers of embedded files if this is not necessary For example data from a 96 well plate assay might be analyzed by 96 different embedded files or by 12 embedded files that each processes 8 sets of data The latter would run far more quickly GF About Page This page records the version number of the GraFit Fitter file It is present for housekeeping purposes only GF Settings Page The entries on this page are used to control the data transfer between GraFit and Excel GraFit Analyses Section Fill in the GraFit analyses section so that there is one entry for each embedded GrarFit file These embedded files can be on any worksheet page with the page name and the name of the GraFit graph being entered into the table TA y 7 8 9 Setting up the GraFit analyses table on the GF Settings page The number of analyses performs dep
54. format 66 67 Text menu 147 Text Orientation 173 Text settings 173 Theory 227 TIFF file 154 Transformation menu Compile command 125 129 Transformations 125 159 179 180 Compiling 129 Curves 128 Data points 126 Defining 129 Error Messages 129 Type Library 315 U Uncompetitive Inhibition 262 Ungroup command 145 Uninstalling GraFit 21 Users Adding 28 deleting 28 V ValidData function 83 84 VBA 309 View menu Grid Lines command 140 Options command 46 Show Outlines command 140 Zoom command 65 141 Visibility Graph objects 167 Index Visual Basic 316 Visual Basic for Applications 309 W Weighting 90 104 221 229 231 Checking 242 Determining weighting type 234 Explicit 246 Window Display Styles 43 Window menu Icon command 40 Windows 2000 46 66 155 Windows 7 46 66 154 Windows metafile 154 Windows Vista 45 66 154 Windows XP 46 66 155 X X Variables 111 113 327 X Y Scatter Graph 134 157 XY graph wizard 157 176 177 178 185 186 Z Zoom command 65 141 Zoom Slider 65 141 341 GraFit User s Guide 342
55. graph frame increasing offset results in the scale being removed from the frame as shown below A No offset x axis offset x and y axes both offset If more than one x axis is present it can be useful to set each to have a different axis offset This allows the two scales to be differentiated Tick marks These settings control the width and color of the pen used to draw the tick marks Major grid Minor grid These settings control the display of grid lines drawn across the axis at intervals determined by the Major increment and Minor increment settings Scale Page LU No grid x axis with major grid solid x axis with major grid solid lines lines and minor grid dotted 169 GraFit User s Guide X Scale Page AY Graph Properties Genera r Jime xn XScale Seale check box for autoscale X ads selector De a E Start 0 Axis extent Entire graph size Finish stat D 1 00 Drawing options Draw Y ads and ticks Scale Type Select linear or logarithmic scaling Graph with linear x axis scale The same graph with a logarithmic x axis scale 170 Chapter 14 X Y Scatter Graphs Scale Start If the box is checked this value is set automatically from the graph data Uncheck the box to specify the starting value of the x axis left edge Scale Finish If the box is checked this value is set automatically from the graph data Uncheck the box to specify the finishing value of the x axis right ed
56. is of the form x Y Y2 Y3 and so should be used when the x data values are common to a set of different y values Multiple Separate X Tab These analyses allow a number of data sets typically up to 8 to be analyzed together and comparison plots made The layout of the data in these templates is of the form x1 Y X2 Y2 X3 Y3 and so should be used where separate x and y values are present for each data set Replicates Tab These analyses are used where there are replicate data sets up to 8 replicates for each data point The data are fitted to the mean of the replicates with error bars drawn to illustrate the spread of the data The descriptions that follow apply to the General case only but can be extrapolated to the Multiple and Replicates cases The files themselves contain instructions on how to use them and should be consulted for more specific information Enzyme Kinetics This template analyzes enzyme kinetic data to the basic Michaelis Menten enzyme kinetics equation Vinax S Km S A graph is made of the fitted data together with an inset Lineweaver Burk plot as shown below 287 GraFit User s Guide Enzyme Kinetics Data 6 D 4 w am 2 0 0 2 0 4 0 6 0 8 1 1 Substrate 0 0 2 4 6 8 Substrate Parameter Value Std Error Vmax 8 2186 0 2298 Km 1 9235 0 1621 IC 50 This fits data to a full 4 parameter equation The binding or inhibition is then characterized in terms of an IC
57. is present that holds a count of the number of items an item in the Values column is present If unchecked that it is assumed that each entry in the Values column is represented once only if checked then the column that holds the number of entries must be selected Start position Enter the starting position for the histogram grouping The histogram records the number of occurrences of data values within a specified data range This range is defined as having an origin at the Start position value and the group size is set by the Increment value Increment Enter the histogram increment value as defined above Set Initial Scaling Histogram Wizard Set Initial Scaling X as Type Linear a Autoscale Manual scaling 0 12 A ands text Y axis Type Linear Autoscale Manual scaling F 1 1 Y ads text Text Number The Set Initial Scaling page allows the scale of the data axis to be specified By default the scale is set automatically and linear axes are chosen These can be altered as required it is Chapter 17 Histograms also possible to adjust the scale settings later The text used for the x and y axes can also be entered Select Tick Styles Histogram Wizard Select Tick Styles OJO E CCU Ls Set the initial tick styles for the graph The tick styles can be modified after the graph is created The Select Tick Styles page allows rapid selection of some of the more common axis
58. is used to show the names and number of the independent x variables that are used in the equation These names are pre defined by the equation library vendor and cannot be edited 327 GraFit User s Guide Ea Hard coded Equation symbol Description ES The X Variables pane of the Hard coded Equation tem Parameters Pane The Parameters pane is used to show the parameters in the equation definition These names are pre defined by the equation library vendor and cannot be edited Hard coded Equation Description Limit Rate constant Offset The Parameters pane of the Hard coded Equation item 328 Appendix 1 Hard coded Equations Constants Pane The Constants pane is used to define constants in the equation definition Constants are used to allow the entry of a value into an equation definition that is fixed in any single experiment but varies from experiment to experiment The presence of constants is optional but when present an equation definition can include any number of constants When constants are included in an equation used for non linear regression their values are entered into the non linear fit item window as described in Chapter 6 a Hard coded Equation General X Variables Parameters Constants Fonts Indents Constants Symbol Description The Constants pane of the Hard coded Equation item 329 GraFit User s Guide 330 APPENDIX 2 GraFit Symbols Font GraF
59. license code Comparison of GraFit Editions Chapter 2 Installation and Activation A comparison of the different GraFit editions is given below Editions Number of users per license purchased Installations allowed Cost per seat Remote license management Remote deletion of licenses Installation password Duration of license License can be renewed Free upgrade to later versions Internet connection required for activation Internet connection needed to run program Serial numbers Single User Licensing GraFit GraFit Academic 1 user per license A single user may install the program on a maximum of two separate computers No No A separate password is used for each user Unlimited Not applicable No Yes No 7OXXXXXXXXXXXXXXXX 7 AXXXXXXXXXXXXXXXX Student Licensing GraFit Student 1 user per license Installation ona single computer only Lowest entry price No No A separate password is used for each user Expires after 1 year No No Yes No 7 SXXXXXXXXXXXXXXXX These installations must only be to support the designated user The first person to install the program provides an email address for someone who becomes the License Administrator This person is responsible for managing passwords etc deletion Multiple User Licensing GraFit Pro GraFit Pro Academic Depends on license size The number of installations allowed is eq
60. linking are extensions of the standard Clipboard cut and paste mechanism Original Cut and Paste Model The original use of the Clipboard was to pass a representation of the object being copied For example copying a graph might place an image of the graph on the Clipboard Pasting would then insert this picture as distinct from inserting the information required to draw the graph Source Application Target Application Standard cut and paste inserts an image representing the source object into the target application This image is simply a picture and contains no information about the underlying data in the source application Embedded Objects Embedded objects hold both a display representation and the underlying data A copy of the current state of the source object is created and is passed to the target application This 211 GraFit User s Guide means that subsequent changes within the source application do not affect the newly pasted object In turn although the embedded object can be edited any changes are not reflected in the original source document Because a complete copy of the source data is placed in the target application files containing embedded objects can be large Source Application Target Application Embedding places a copy of the internal data into the target application as well as an image representing the source Linked Objects Linked objects also copy a representation of the original data b
61. made The result of the comparison is either 0 if FALSE or 1 if TRUE To see how these operators work consider a graph of the form y x lt test 1 3 Here x is the x axis and test is a constant that has the value 2 This function is plotted below 117 GraFit User s Guide These comparisons can be used to introduce a discontinuity into a curve and though they will seldom be used in equations are extremely useful for displaying limits etc to annotate a graph An alternative method to obtain discontinuities is to use the If Then Endif constructs described below Note To obtain a sharp discontinuity it is necessary to create the curve with Resolution set to High see page 163 Numbers can be entered directly into the equation definitions and can include exponents e g 2 8 e 12 Functions A large number of functions are provided for use in equation definitions Function Returns abs x Absolute value of x i e if x lt 0 returns x otherwise returns x ascos x Cos of x alog x Antilogarithm to base 10 of x asin x Sin of x atan x Tan of x cos x Cosine of x exp x e LEAC The fractional part of x The fractional part of 13 387 is 0 387 int x The integer part of x The integer part of 13 387 is 13 log x The logarithm to base 10 of x 118 Chapter 9 Equation Item Function Returns ln x The natural logarithm of x Sin x Sine of x sqr x The square of x x
62. no lines are drawn Click the Finish button to create the chart 205 GraFit User s Guide 206 CHAPTER 17 Histograms The Histogram Wizard Chapter 12 described the basics of creating graphs Selection of Histogram produces the Histogram Wizard to assist with the procedure all settings can be adjusted later if required The process of editing histograms is broadly similar to editing X Y graphs and Chapter 14 should be consulted for further information Select Data Source Page Histogram Wizard Select Data Source Legend text Data set 1 SOUrCe Values column E Numbers column start Increment You can use the Graph Add Data command to add more data sets to the plot Cancel The Select Data Source page allows you to choose the source and type of data to plot Legend text The data set plotted is associated with an entry in the data legend The entry shows the symbol used to represent the data and a descriptive text The initial text used to describe the entry can be entered into this field 207 GraFit User s Guide 208 Source Graphs are plotted using data that are present in one of the items found in the GraFit workspace The Source selection lists all items that contain suitable data for plotting and one of these must be selected Values column Select which column holds the data values whose distribution is to be plotted Numbers column Check this box if a second column
63. no physical significance The choice of equation will depend upon the shape of the data and it is possible that more that one equation would prove suitable In the case shown above it might be appropriate to use a polynomial or any equation that can produce a gentle curve Below is shown the results of fitting the data to a second order polynomial y a bx cx using a Linear Fit item and to a first order rate equation by non linear regression The latter equation has no possible theoretical justification but has a suitable mathematical form to allow a smooth curve to run through the data 1 2 1 2 g 08 08 F E o 0 6 Ss 06 YN Jp lt lt 0 4 0 4 0 2 0 2 0 O 0 O O 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 Concentration Concentration 255 GraFit User s Guide Reading Values from the Fitted Curve Either of the fitted curves shown above will allow the concentration values that correspond to a series of measured absorbance values to be read from the curve This is performed by entering the unknown absorbance values into a data column then generated the calculated values in iS column using the Column Type gt Calculated Values x Values command Table tab Calculated X Values Fitting results and order polynomial Unknown Absorbances Estimated from polynomial Estimated from non inear fit Interpolate between X limits From 10 To 200 Fitting Several Data Sets
64. on CD ROM the activation code serial number will be present on the software case if you have downloaded the software you may purchase an activation code online During activation you will be asked to provide a password which will be needed in future if you reactivate or deactivate the software You will also be asked to provide an email address This address is used to allow automatic reminders to be sent if you forget your password Activation is done via the internet if your computer is not connected to the internet it is possible to carry out offline activation See the online help for a full description of how offline activation is carried out Chapter 2 Installation and Activation Deactivating GraFit Your GraFit software needs to be activated on line during which time a count is made of the number of times an individual activation code is used You will not be allowed to activate software on more computers that is allowed by your license agreement This means that if you wish to move your software to a different computer you should first deactivate it on the original computer Note Once you have deactivated your software it will no longer run until it is once more reactivated Deactivation To deactivate your previously activated software select the Help About dialog and select the Deactivate button You will need to enter the password that you gave when you originally activated the product If you have forgotten your password
65. program has been activated it will run without showing the activation dialog box again Activation Dialog Box Before GraFit has been activated running the program will produce the following Software Activation dialog box Grafit 7 Software Activation Activate GraFit 7 Mar TA Activation Code full or evaluation WI LAI EL Evaluate GraFit 7 Click the Evaluate button to obtain an evaluation code Evaluate Software Activation dialog box This software is not yet activated To activate the program enter your Activation Code either full or evaluation into the box above and click the Activate button To evaluate GraFit during the trial period click the Evaluate button Change Proxy Settings Purchase activation code online 18 Chapter 2 Installation and Activation While the program is being evaluated this dialog box shows the remaining evaluation period if you are still within the 14 day evaluation limit then you can run the program by clicking the Evaluate button Once the evaluation period has expired you will not be able to run the software until you have entered a full activation code Evaluate GraFit 7 I During the evaluation period the You have 5 days of your evaluation period a UN UA number of days remaining is remaining Evaluate gt shown Click Evaluate to run the f program Setting Proxy Settings For some users access to the internet needs to be via a Proxy Server Providing you can
66. than another This test can be used to ascertain whether it is realistic to use an equation with additional parameters over a simpler version or whether one equation fits a data set significantly better than does a second For example the analysis shown below fits an exponential decay to a single exponential equation y Ae In addition it fits the data to an equation that has an additional background parameter y Ae background A sample set of data fitted to each of these equations is shown below 73 GraFit User s Guide 74 120 Experimental data 100 Fit Single exponential 80 Fit Single exponential 4 with offset E 60 lt 40 20 0 0 1 2 3 4 5 6 Time Using the F test it is possible to test whether the data are fitted significantly better by the presence of the extra parameter By eye the fit obtained with the additional background parameter seems slightly better than the one without but this is subjective and it is better to use a statistical test Chapter 5 Data Tables F Test Fitting results 1 Fit Single ha Cancel Fitting results i Fit Single Offset 1 2 3 4 F Diference Fa Time Amount 0 0000 110 0000 1 3654 Analysis 1 1 0000 80 0000 1 4794 Fit Single 2 0000 60 0000 1 1121 Single Exponential Decay 3 0000 45 0000 0 8360 Number of parameters 2 0000 4 0000 35 0000 0 6216 5 0000 28 0000 2 2151 Analysis 2 Fit Single Offset single Expone
67. the column type header The difference is in the arrangement of data columns For this layout pairs of x y data columns are arranged contiguously in the data table and a separate set of x data values are used for each y data set All the x data columns hold values corresponding to the first independent variable as defined in the equation values for the second independent variable are obtained from the type header The First X data column and the Last Y data column are selected and from this the program can work out how many data sets are present xn fromtable a e This layout is available whenever there is more one independent variable and is the only layout type when three or more independent variables are present When using this layout the program determines which column is associated with which independent variable by examining information present in the column type headers of the data table The column types of the various independent data sets x axes need to be set to x n Data with the n value representing the order of the x variable found in the equation definition See Chapter 5 for further details on column types The position of the y data column is selected from the Y data column entry X Data Column This entry specifies the column that holds the x data values See the Data Layout section above for further information 92 Chapter 6 Non linear Fit Item Limiting X Data Range It can s
68. the mouse on the column numbers to select entire columns of data Column resize region Click and drag the edges of a column to resize it Row selection region Click and drag the mouse on the row numbers to select entire rows of data within the data table Row resize region Click and drag the edges of a row to resize it t Split table horizontally Drag the splitter box on the horizontal scroll bar to divide the data table window in two The two halves can be independently scrolled allowing distance regions of the table to be viewed simultaneously p Split table vertically Drag the splitter box on the vertical scroll bar to divide the data table window in two The two halves can be independently scrolled allowing distance regions of the table to be viewed simultaneously Resizing Rows and Columns The row height and column width can be set using the Row Height and Column Width commands on the Table tab respectively or by using the mouse to drag the edges of the rows or columns within the row or column selection region Columns can be sized to fit their contents using the Size Column to Fit command on the Table tab 17 The Edit Edit Current Cell command in legacy mode The Table Row Height and Table Column Width commands in legacy mode The Table Size Column to Fit command in legacy mode 18 19 54 Chapter 5 Data Tables Data Types The number of data points that can be entered is limited onl
69. tick styles More extensive tick style choices can be made once the graph has been created 209 GraFit User s Guide Select Bar Style Histogram Wizard Select Bar Style Fill style Type Bar type Ol ia Fill colors oS Fore SMEA Back Outline style Width 08pt E a The Select Bar Style page allows the histogram bar representation to be chosen Click the Finish button to create the chart 210 CHAPTER 18 Embedding and Linking What is Embedding and Linking Embedding and linking are two related ways that information from one application can be incorporated into a different program Also known as Object Linking and Embedding OLE it provides a uniform mechanism for Windows programs to cooperate and to share information Most Windows applications allow information to be copied between programs using the Clipboard For example a picture could be copied from one application and pasted into a second The problem with simple cut and paste is that the second application often has no idea what information is being transferred In non OLE applications this often means that once the information is copied it is impossible to make any subsequent changes to the image OLE gets around this by allowing two cooperating applications to pass information between themselves in such a way that editing is still possible even after pasting into an application that has no knowledge about the information transferred Both embedding and
70. to 2 of the delivered volume and the so error is proportional to the recorded value Individually determined error All the above cases make assumptions about the error of each measurement However if sufficient replicates of each data point are available or a complex error function is applicable it is possible to calculate os for each point and use these values explicitly This method can also be used if you know that certain values are less accurate than others for example if they were measured in a different way as again only relative gf values need be given In practice the use of simple weighting or proportional weighting is most often appropriate If the errors are small the weighting function chosen has only a small effect on the calculated values We will see later how it is possible to estimate which type of error function best describes a set of data If any data values are close to O it is not appropriate to use proportional weighting and simple weighting should be selected Non linear Regression 232 For an equation that is not a linear function the determination of best fit parameters becomes slightly more difficult although the principles discussed above still apply The difficulty arises because it is not possible to derive an exact analytical expression for minimizing Y It is possible however to take initial estimates for the best fit parameter values and to improve on them by a series of iterative calculations unti
71. value can be described as a function of the x data and a series of parameters i e y f x Py Po P3 As a result it is applicable to many more situations than linear regression and is more generally useful for analyzing experimental data The only drawback to non linear regression is that the calculations are too complex perform using a calculator and require a computer program such as GraFit Additional Considerations for Non linear Regression The more complex nature of the non linear regression calculations does require some further considerations compared with linear regression Equation to Use Non linear regression can employ a vast range of different equations It is therefore necessary to select an equation that is appropriate to the particular experimental situation before fitting the data Initial Estimates The mathematical method used to perform non linear regression acts to take starting estimates for the parameters in the equation and optimize them during the calculations As described in Chapter 9 GraFit provides a mechanism for providing these initial estimates Chapter 19 Data Fitting Basics automatically for some equations but in principle it is necessary to give the calculations some rough estimates prior to data fitting Convergence Criteria Unlike linear regression non linear regression does not provide an exact solution but by an iterative process calculates successively better parameter value
72. value using the following equation Range O RES background ae 7 A graph is made of the fitted data as shown below 288 Chapter 23 Standard Templates IC50 Data 100 80 o Y O D qa 60 pr 40 20 10 10 10 10 10 Inhibitor Parameter Value Std Error Y Range 79 0926 6 9937 IC 50 1 47142e 007 3 99433e 008 Slope factor 0 7194 0 1517 Background 26 0055 4 5106 IC 50 Back Corrected This fits data to a version of the IC equation that pre supposes that the data have already been background corrected The binding or inhibition is then characterized in terms of an IC value using the following equation v Range TNS 1 E A graph is made of the fitted data as shown below 289 GraFit User s Guide IC50 Background Corrected Data 400 0 U a O Q U l or 200 0 10 10 107 10 10 Inhibitor Parameter Value Std Error Y Range 407 1703 6 5733 IC 50 5 46427e 007 5 51388e 008 Slope factor 0 6397 0 0355 Ligand Binding This template analyzes ligand binding data to single site saturation equation L Cap Ka L A graph is made of the fitted data together with an inset Scatchard plot as shown below 290 Chapter 23 Standard Templates Single site Ligand Binding 12 10 8 Oo gt a 6 4 z 3 2 6 8 10 12 Bound 0 0 20 40 60 80 Free Parameter Value Sid Error Capacity 17 3459 0 6126 Kd value 29 7734 2 5451 Linear Regression This template al
73. with Some Parameters Common and Some Specific 256 To illustrate imagine that you have collected two linear data sets and that you already know that for this experiment the slope is the same for each although the intercepts are different Using GraFit it is possible to fit these two data sets to find the best slope that is common to each and the two intercept values that are specific to the two data sets Indeed it is possible to fit two or more data sets to any of the normal equations that GraFit can use and to share one or several parameters between the data sets The example below shows how this is done for the paired linear data Two raw data sets are illustrated below these data are in the example file Paired linear regression Fitting these two data sets individually results in similar but not identical values for the slope This is shown in the figure below Chapter 21 Worked Data Fitting Examples Fit to common slope O y1 slope 1 975 y2 slope 1 975 If the data sets are known to have the same slope this can be incorporated into the data fitting as follows Method Used to Share Variables between Data Sets Sharing variables between data sets involves using an equation with a second independent x variable which is defined in such a way that that this value acts as a switch to decide which parameters apply to the data value For the simple example described above the independent variables are
74. writing brief documentation on how to use a particular data file or template The notepad item is created from the main GraFit window using the Add Notepad command The Notepad Item Window 4 Motes Hello These notes are stored in the GraFit workspace Notes can e text with MULTIPLE fonts stiles Sie and colors They can also include embedded objects such as the Microsoft Paint picture added below The Notepad item window The font styles are set from the Font tab Notepad windows automatically wrap their contents so that the line width is equal to the width of the window Alternatives to the use of Notepad Items The notepad item provides a simple text editor that has many of the capabilities of the Windows WordPad application However it does not provide the wealth of features present in a dedicated word processing application such as Microsoft Word It is however possible to The Font menu or the formatting toolbar in legacy mode 131 GraFit User s Guide harness the power of applications such as Word by embedding documents into the GraFit workspace For example a Word document can be embedded within the main GraFit window as described in Chapter 18 Incorporating notes in this manner can be a more suitable alternative to using a notepad item 132 CHAPTER 12 Creating Graphs How to Create a New Graph To create a new graph first add a graph item window to the workspace using the Add Grap
75. x and dataset parameters al and a2 are the two intercepts and b is the common slope The equation is then defined as If dataset 1 Then Vee ee lo ax Return Eo de a2 b x How does this work When data from the first data set are accessed the value of dataset is 1 and the equation returns at t pz When data from the second data set are accessed the value of dataset is 2 and the equation returns a2 b x Therefore the value of a1 is determined in effect from only the first set of data and a2 from the second However the slope b is applicable to all the data The graph below shows these data fitted to a common slope in this manner 257 GraFit User s Guide Fit to individual slope O y1 slope 1 937 y2 slope 1 970 The dataset setting is set using the Column Type Data Types gt y Data with Associated Value command Table tab resulting in the appearance shown below 3 xa 1 0000 x 2 0000 y1 y2 1 0000 2 0000 3 0000 4 0000 5 0000 6 0000 7 0000 6 0000 9 0000 10 0000 3 9814 5 3816 6 1534 10 5019 12 0741 13 0814 15 7954 17 4193 19 4161 22 0609 6 7743 8 5039 11 2915 14 1558 14 5192 17 0320 18 5655 21 0429 22 9451 24 6848 It is simple to extend this approach to many data sets The equation to find the best slope through four data sets and a separate intercept for each is If dataset 1 Then al b x Return EIA 258 Chap
76. 05143 Km 2 0694 0 3724 aFit Source ln Destination Excel mn Non linea 2 Data D30 Pi gt pp _ _ _ 443 6 7 nae e Fit Data 4 10 Data Analysis 12 1 Ensure that the table on the GF setting Yara aa landing data fomJinkad fii Chapter 24 Passing Data Between Applications Incorporate the data to be fitted into this file either on the blank initial worksheet page as here or by adding additional worksheets Embed the GraFit graph into the file Setting up the analysis involves making entries on the GF settings page to define the way the data transfer is done On the GF settings page specify that the GraFit data fitting object is called in this case GraFit 1 and is on the Data page that the data to be fitted is also found on this page and occupies the cells A1 to B31 The data is inserted into the GraFit object s Data data table starting at column number 1 Note that the top line of this data region is used to provide the column headings the remainder provides the data to be fitted On the GF settings page specify that the results are held in the GraFit item called Non linear Fit that there is a single fit and that the analysis involves 2 parameters These results will be transferred back to Excel and placed into worksheet page Data starting at cell position D30 To fit the d
77. 110 0000 Initial value 108 6349 J Value name 1 0000 60 0000 Rate constant 0 2676 2 0000 60 0000 3 0000 45 0000 4 0000 350000 5 0000 280000 g 10 ai 4 F After the fit to be displayed is selected the parameters available are listed Some or all of these can be included by clicking in the check box to the left of the parameter name If more than one set of data were analyzed in the selected fit the results for each fit are given It is possible to display either the fitted parameter value or the fitted standard error value as chosen in the Display settings In addition the name of the parameter can optionally be displayed next to the data value by checking the Value name option 76 Information Chapter 5 Data Tables This column type lists information about a data fitting item Data Fitting Information Fit Fit Single Fitting information JA data column name 4 UY data column name Y Number of data points J Chit value Reduced Chi value Data Column 4 Display value name ate E to ha a LD co ok Cancel Ed me ay 1 2 3 E Fitno Initial Rate 0 0000 110 0000 X column Initial 1 0000 60 0000 Y column Rate 2 0000 50 0000 data 6 0000 3 0000 45 0000 Chi 11 2814 4 0000 35 0000 Reduced Chi 2 8204 5 0000 28 0000 After the fit to be displayed is selected the available information items are listed Some or all of these can be included by clicking in the check box to the
78. 2 If the fits are equally appropriate this ratio will be 1 0 the F test assesses the significance of the observed differences Mean and Standard Deviation For a full description of means and standard deviations see any text on statistics for example that by Bevington A brief description follows The mean value of a set of data points is defined as _ 1 wxk x Here u is the mean of the parent distribution and x is the mean of our experimental sample The uncertainty or standard deviation of the data points o is estimated from the data as follows The uncertainty of the mean Ow is given by the equation O S O Fe VN VN All the above equations assume that the uncertainties of all the data points are equal If they are not for reasons described earlier we must use equations that incorporate weighting For example calculation of the mean becomes d x 07 1 07 exe 236 CHAPTER 21 Worked Data Fitting Examples This chapter provides some further examples of data fitting to help acquaint you with the program The examples are presented so that they can be worked through in a step by step manner The data files that are used can be found in the Samples sub directory of your My GraFit Data directory Don t worry if the examples do not seem relevant to the type of data that you need to analyze the same principles are involved in all data fitting This chapter assumes that you are
79. 28 Regression analysis is probably the most powerful method to analyze data However such analysis involves several assumptions about the function chosen and the error structure of the data These assumptions are listed below The Correct Equation is used This assumption is often overlooked but should be borne in mind when using any data analysis technique It is important to be aware that deviations from ideality are often caused by use of an inappropriate equation Graphical methods provide a useful means to see whether the chosen model equation is appropriate This is because use of an inappropriate equation often results in systematic deviations from the curve which is easy to see in a graph Residual plots of Ay versus x are useful to assess whether the equation is appropriate Systematic deviations in the residuals can mean that the equation used was inappropriate to describe the data The differences between experimental and calculated values can be calculated using options available in the data table see Chapter 5 The data shown below have been fitted to a single exponential decay and to a double exponential decay and the residuals from each have been plotted Clearly the residuals from the double exponential fit are smaller and more randomly distributed and show that this fit is superior Chapter 20 Theory 40 Errors Double Exponential
80. 42 49 238 242 Adobe Acrobat 18 Align command 145 Allosteric Data 246 Allosteric Kinetics 270 Arithmetic operators 117 Arrows 152 ASCII format 66 67 Automation Reference 315 Axis break symbol 196 Axis manager 190 192 193 Axis offset 169 Axis scaling 195 B Bar charts 136 199 Bezier curves 165 Bitmap file 154 Bring to Front command 146 C Calculate menu 333 GraFit User s Guide Fit Data command 103 240 Calculated values 78 Calibration Curve 254 Changing Computers 21 Chart Wizard 199 Chi 96 Chi squared 227 230 Clear command 39 61 62 Clipboard Windows 213 214 215 Clipboard 211 212 Column Combinations 71 Column charts 133 135 199 Column Combinations 71 Column constant 83 Column Format command 63 Column type 67 A B 68 71 A B 71 A B 71 245 A B 71 Calculated Function 82 252 Column Summary 72 Constants Entry 78 Derivative 85 Difference 82 243 F Test 73 General 69 Information 77 Mean 71 No Data Points 72 Parameter Entry 77 Results 76 252 Series 85 Smooth 86 Standard Deviation 72 Standard Error of the Mean 72 Subset 86 x Data 69 334 x Values 79 254 256 x n Data 69 y Data 70 y Data with Associated Value 70 258 y Values 81 254 Column Width command 54 Columns Copying and pasting 59 Deleting 62 Selecting 59 Comment statements 120 Competitive Inhibition 260 262 Compile command 109 12
81. 5 Constants 93 114 162 185 329 Linking to Data Table Entries 93 Context Help 43 Cooperative binding 278 Copy command 59 60 212 215 Copy To command 66 CSV format 66 67 Cursor shapes 54 144 Customize button 36 Customizing Menus and Toolbars 37 Cut and paste 211 D Data Association 194 Copying 59 Deleting 61 Export 66 Fitting several data sets simultaneously 256 Import 66 Import Export 66 Inserting values 62 Layout 90 104 Missing 55 Numbers 55 Overlaying 189 Pasting 59 Rescaling 82 Selecting 58 Smoothing 86 Sorting 63 Text 55 Types 55 Data entry position 53 54 57 59 66 68 95 Data Filters 49 Data fitting Basics 217 Multiple Non linear Regression 224 Data Fitting 75 Data function 83 Data table Appearance 63 Locking columns 87 Data table item 53 Deactivation of GraFit 21 28 Definition 115 Degrees of freedom 227 Delete command 62 Denaturation Curves 259 Derivative 85 DIF format 66 67 Difference calculation 67 Digitally Signatures 298 Directories 47 50 Dixon Plot 261 Dose response Curves 273 Double exponential decay 272 Drag and drop 39 Draw menu Results Listing command 98 107 240 Drawing Objects 140 Index E Edit Current Cell command 54 Edit menu Clear command 39 61 Copy command 59 212 215 Copy To command 66 Edit Current Cell command 54 Hide Object command 167 194 Insert New Object com
82. 89 GraFit User s Guide 100 80 0 C a 60 Axis split T oc 40 20 10 10 107 10 10 104 10 Inhibitor Creating and Managing Multiple Axes By default GraFit graphs have just a single x and y axis Any additional axes must be added to the graph using the Axis Manager which allows axes to be created or deleted and simplifies generation of split or multiple axes Running the Axis Manager To activate the Axis Manager use the mouse to select the graph that you wish to modify then choose the Axis Manager command from the Graph tab This will launch the Axis Manager Wizard The Graph gt Axis Manager command in legacy mode 190 Six options are available on the initial page Axis Manager Choose An Option Select an option T Add a new x axis I Split an existing x axis M Remove x axis Add a new x axis Add a new y axis Split an existing x axis Split an existing y axis Remove x axis Remove y axis Chapter 15 Using Multiple Graph Axes If only a single axis is present the Remove axis options will not be available as a minimum of one axis must exist Select one of the options and click the Next button Adding a New Axis Choosing to add a new axis will give the following page 191 GraFit User s Guide Axis Manager dd y Axis Initial tick style Houuoo Text location Axis offset E Ro A number of initial tick styles and axis text locations can be chosen and i
83. 9 0 05 V 0 055 0 005 C 0 7 0 5 Displaying Multidimensional Data Displaying data sets that have more than one x axis can be difficult One solution is to remove all but one dimension from a plot For example in the above data a plot of logk V Vol versus pK will result in a linear graph with a slope B and intercept c We can create the logk V Vol data using the data manipulation abilities of GraFit the result for Vis obtained from the curve fitting The first step assuming that the data have already been fitted is to allow the calculated V value to be accessed This can be done by creating a data column that displays the fitting results To do this select a blank column and choose the Column Type gt Data Fitting gt Results command Table tab and select the appropriate Non linear Fit item The second step is to select the next blank column and choose the Column Type Data Processing Calculated Function command to set up a calculation The following calculated function allows the column to be filled with the logk V Vol entries Table Column Type Data Fitting Results command in legacy mode Chapter 21 Worked Data Fitting Examples A Calculated Function Number of Rows 5 Number Result log data 3 row Data fitting results Log Rate V Vol 1 column s selected Note the use of the Data function to access data present in the data table The appearance
84. 9 for equation definitions although the definition is limited to a single line and only the right hand side of the Definition equation is entered The definition can be of arbitrary complexity and can include mathematical functions parameter names etc The pre defined symbols xdata and ydata represent the original x and y data values respectively The symbol xdata2 can be used to access any data values associated with the y data column y Data with Associated Value column type see the discussion of column types in Chapter 5 In the example illustrated above we wish to take reciprocals of these data the definitions are therefore 1 xdata and 1 ydata Any algebraic expression may be entered for example xdata ydata sqrt xdata is a perfectly valid transformation definition It is permissible to access the values of parameters and constants in the equation if desired The symbol names of these are listed at the bottom of the Data Points pane 127 GraFit User s Guide Generating Residuals Plots A very useful symbol that is specific to the data transformation definition is ycalc This returns the y value that is calculated from the fitted parameters Therefore to generate a residuals plot define the transformed y data as ydata ytale Axis Name Settings When first creating a transformed plot the text used to name the x and y axes is taken from the Default X name for graph and Default Y name for graph entries T
85. Data bar ar ET A 7 AA AA A SS E al q El 1 2 gt Data GraFit Untitled oO rel x V Options 69 G NA 3 X Select All LG Add Stock Item ii fal Edit Text d aste do Redo Cut Copy Clear l Store Stock Item Black fi Text Strings EH Notes fifty Graph 2 Py Enzyme Kinetics Data Instructions A ae po pet A AI AS sFoiopenbraa fo adap im tbe f m dm gt GraFit Untitled Pia Options amp gt 3 Import Data do E gt Select All baal GA Add Stock Item E Edit Text 4 Paste Undo Redo Copy Clear Si ay Store Stock Item Items Clipboard Edit Object S I I ver Text Strings Fa Notes i aes fi Graph BX Ed Notes E nomea aso Data Instructions B Ben geen IS mnt ape o 2 View gt Style command in legacy mode 43 GraFit User s Guide G amp Import Data ay Add Stock Item bg Store Stock Item View Soa y Paste Undo Redo Cut Copy Import Data aj Add Stock Item y Say Store Stock Item Clipboard 44 Clear GY Select All fall Edit Text Enzyme Kinetics Data e sl a GY Select All E Edit Text Graph 2 Object Enzyme Kinetics Data cd O X Notes 2 Instructions toa nate arate q Options O x o a 2 E Notes Instructions nter substrate Aqua Scenic Windows 7 style White Office 2010 style CHAPTER 4 Working with Files GraFit Workspace Files The GraFit workspace consists of all the items displayed in the m
86. E ji only in a horizontal direction or only in a vertical direction respectively ry The cursor is over a selected object that can be moved in any direction f and the CTRL key is down This means that dragging the element s will result in creation of a copy of the selection Polygon objects only The cursor is over one of the lines of the polygon and the CTRL key is down Under these circumstances left clicking will add an additional control point to the polygon e Polygon objects only The cursor is over one of the control points of the polygon and the CTRL key is down Under these circumstances left clicking will delete this control point from the polygon i Text objects only The cursor is over the text object which is currently open for editing Grouping Objects Drawing objects can be grouped in order to ensure that they can be moved as one To group objects select those required and choose the Group command found in the Objects group of the Graph tab Objects that have been grouped can have their grouping removed using the Ungroup command Aligning Objects Selected objects can be aligned by selecting those required and choosing the appropriate Align command found on the Graph tab Objects are aligned relative to all currently selected gt The Object Group command in legacy mode The Object Ungroup command in legacy mode The Object Align command in legacy mode 59 60 145 GraFit U
87. Error of the Mean Each row entry in the column is set to be the calculated standard error of the mean for the corresponding values from the columns selected in the above dialog SEM o is defined as O O H VN Column Summary The entries in the column give a summary of the values present within a single selected column Chapter 5 Data Tables Column Summary Statistics Source data column Value 1 Cancel Value 3 Column 4 Data 1 2 3 A A Type Summary sd Walue 1 Value 2 Value 3 4 3000 2 1000 2 2000 Source Value 2 3 5000 4 6000 1 1000 Data points 5 0000 6 2000 5 6000 0 6000 Minimum 2 1000 4 4 6000 3 6000 1 0000 Maximum 5 6000 5 4 0000 3 9000 0 1000 Range 3 5000 6 Total 19 8000 T Mean 3 9600 G Median 3 9000 3 Variance n 1 3384 10 Variance n 1 1 6730 11 Std dev n 1 1569 12 Std dev n 1 1 2934 i This summary gives the number of data points minimum and maximum data values the data range sum mean median variance calculated for a divisor of n and of n 1 and standard deviation calculated for a divisor of n and of n 1 F Test The entries in the column show the results of performing an F test comparing the goodness of fit of two fitting analyses that use different equations A fuller description of this test is found in the theory section Chapter 20 but briefly the F test evaluates whether one fit is significantly better
88. FF 45 File menu 35 38 Add Stock Item command 42 49 238 265 Close command 49 Export Graph command 154 Import Data command 49 66 336 New command 42 50 51 285 Open command 45 238 246 Page Setup command 41 139 Print command 40 97 106 Print Preview command 40 97 101 102 106 240 Save As command 48 50 Save command 48 Store Stock Item command 42 49 Files Recently Used 47 Creating new 49 Exporting graphs 154 Importing Data 49 Opening 45 Pinning to the File List 48 Saving 48 Saving Legacy Versions 48 Template 49 Filters Import Export 49 Fit Data command 40 89 240 Fitting criteria 96 Font selection 100 101 Font settings 107 131 153 154 Footers 42 For statement 121 Formatting numbers 63 99 172 Formatting Options 101 Frequently asked Questions 31 Functions 118 G GF_module bas 313 320 Global Analysis 223 Goto Statement 121 GraFit Installing 17 Running 35 Technical Support 15 Templates 42 Uninstalling 21 GraFit Academic 22 GraFit Student 22 GraFit Editions 22 23 GraFit Fitter Excel Template 300 GraFit Fitter Reference 302 GraFit Items 39 GraFit Pro 22 GraFit Pro Academic 22 GraFit Workspace 38 45 Graph Axis associations 186 X ticks 168 Graph Magnification 142 Graph menu Add Data command 186 242 Axis manager 190 New Graph command 133 Preferences command 140 147 Graph types 133
89. Fit Data Search File name Save as type Text Files a Browse Folders The Save as type entry determines the format in which to save the exported data GraFit supports a number of data export formats and the available types are listed in this box GraFit supports export as ASCII Text DIF and CSV formats as standard though additional formats may be available Windows 2000 and Windows XP Earlier versions of Windows use the older style file save dialog box 3t The Edit Paste From command in legacy mode 32 The File Import Data command in legacy mode 33 The Edit gt Copy To command in legacy mode 66 Chapter 5 Data Tables Copy To File Save in 900 E GraFit Data My GraFit Data my Music my Pictures My Videos My Documents My Network Places a GraFit Data EJ Perm GraFit Templates Save as type Text Files The Save as type entry determines the format in which to save the exported data GraFit supports a number of data export formats and the available types are listed in this box GraFit supports export as ASCII Text DIF and CSV formats as standard though additional formats may be available Column Types Columns can hold simple data values or can be set to hold the results of various calculations or data manipulations For example a column can hold the difference between the values in two other data columns The results of these calculations are automatically upd
90. GrarFt User s Guide Version 7 Kj Import Data y DC X Select Al a 3 GA Add Stock Item Ga Edit Text Add Paste Undo Redo Cut Copy Clear Object 3 Store Stock Item Edit Object 0 02040603 1 1 Substrate 4 e Substrate Parameter Value Std Error Vmax 9 5250 0 4458 Km 2 5114 0 3092 m l 1 Enter substrate rate data into the data table 2 Watch RO Erithacus Software Erithacus Software GraFPit User s Guide Version 7 Erithacus Software Limited 1989 2011 Program and Manual Designed and Written by Robin J Leatherbarrow GraFit User s Guide Copyright Copyright Erithacus Software Limited 1989 2011 All rights reserved Portions of the software described in this document copyright Microsoft Corporation 1985 2011 All rights reserved Information in this document is subject to change without notice and does not represent a commitment on the part of Erithacus Software Limited The software is furnished under a license agreement and the software may be used or copied only in accordance with the terms of the agreement The purchaser may make one copy of the software for backup purposes No part of this publication may be reproduced transmitted stored in a retrieval system or translated into any language in any form or by any means including photocopying and recording for any purpose other than the purchaser s personal use without the writt
91. H Number of data points A es The Results pane of the Non Parameter Je linear Fit item If more than one set of data is specified in the Settings pane each fit can be viewed by clicking the arrow buttons The File Print and File gt Print Print Preview commands display the results as illustrated below 97 GraFit User s Guide G GraFit Untitled A XA oa Prit gt 4 0 A a Close Preview Untitled Non linear Fit 07 November 2007 01 22 in Leatherbarrow Enzyme Kinetics jei i Simple athena na aa ag Using Print Preview from the Data table Data gt Fy ren va Non linear Fit item Results X data column Substrate Y data column Rate Number of data points 5 Initial estimates From equation definition Vmax 7 6068 1 6490 Km Number of iterations 2 Reduced Chi value 0 0932 Parameter Value Std Error Vmax 3 6146 0 7578 1 6700 0 4616 The formatting of these results is controlled from the Number Format Fonts and Indents panes Showing the Fitted Results on a Graph A listing of the fitted parameter values can be displayed in a graph item window by selecting the Results Listing command on the Graph tab See Chapter 12 for further information 2 The Draw gt Results Listing command in legacy mode 98 Chapter 6 Non linear Fit Item Number Format Pane The numeric result values are displayed formatted as is specified on the Number Format pane Ea
92. Ka L Ka1Ka2 L Kar Kaz Kan background In this equation B is the amount bound L the concentration of free ligand Cap the maximum capacity of the system to bind ligand and K K etc the successive dissociation constants for ligand 2 Adair 3 sites Use this equation for 3 binding sites 2 Adair 4 sites Use this equation for 4 binding sites 277 GraFit User s Guide pH 278 2 Bound vs Total 10 The previous equations require the ee unbound ligand concentration to be 67 known However although the total 5 4 Bling Carus ligand concentration is always known 2 da lo this is what you have added the proportion bound and free is not always UA DA E e e certain This is particularly the case if the Total y axis is an observation that is not directly related to the bound concentration for example fluorescence intensity or absorbance Here the equation relates the y axis to the total ligand added t Ka t Cap y Ka t Cap 4 t Cap T Bound vs Total tight binding limit This transformation shows the maximum possible amount bound or change in absorbance etc for a given total amount of ligand added if the binding were infinitely tight 2 Cooperative A generalized equation describing cooperative binding or multiple binding sites of differing affinities is _ L Cap K L Here n is a measure of the co operativit
93. LO 333 12 CHAPTER 1 Introduction Welcome GraFit version 7 for Microsoft Windows combines presentation quality scientific graphs with comprehensive data fitting The result is a powerful package that is easy to use produces stunning output and can also fulfill the needs for quantitative data analysis Data can be fitted using various types of theoretical models and your own equations can easily be added to those supplied with the program Comprehensive graph plotting facilities allow you to display your data on screen and edit the graphs interactively Key Features of GraFit GraFit provides the experimental scientist with a tool for visualization and analysis of their data For the occasional user GraFit can be configured to perform routine analyses with minimal intervention in many cases it is possible just to enter the data and allow GraFit to calculate the results and plot the graphs in the background Advanced users can utilize the full power of GraFit for analyzing complex equations and may create custom fitting templates for routine data processing 13 GraFit User s Guide About this Guide This manual describes how you can use GraFit to examine your data and to plot graphs We recommend that you read the manual fully and try out some of the examples described Notational Conventions The following notational conventions are used throughout this manual Convention SMALL CAPITALS Monospace Bold Me
94. Non linear Fit Settings Constants Estimatrix Fitting Criteria Results Number Format Fonts Indents Decimal places Max digits 12 Thousands separator Scientific format Y The Number Format pane of the Non linear Fit item J Use scientific format for small values Trailing zeros in fraction Specification Decimal places Max digits Thousands separator Scientific format Use scientific format for small values Show 0 s Meaning Enter the number of decimal places to be displayed for numbers in the results listing Note To ensure that all decimal places are displayed set Trailing zeros in fraction to Show 0 s Enter the maximum number of digits to use when displaying numbers in the results listing Check this box to include a grouping separator The symbol used for this separator and the number of digits in the group are set for the View Options command on the Home tab Check this box to display the values using scientific format e g 2 435e003 instead of 2435 000 Check this box to ensure that small values which would otherwise be displayed as 0 0000 are shown in scientific format e g 2 0987e 008 99 GraFit User s Guide Specification Meaning Trailing zeros in Chooses the way that trailing zeros are displayed in the formatted fraction numbers It is possible to show the O characters to omit them or to replace them with spaces Fonts and Indents Pan
95. S In this equation Vaa and K are the parameters that we need to obtain from data fitting The usual way of analyzing these data is to collect a series of progress curves A as a function of t at varying concentrations of substrate For each concentration of substrate the data are fitted to the first equation and a value for k obtained The resulting k values are then fitted to the second equation to find Vmax and Km A better solution is to recast the equation as a three dimensional one where the progress of the reaction varies with t and S which are the two independent variables The equation is obtained by combining the previous ones At Aw E exp ta This allows Vmax and Km to be determined directly from the raw data which has two x variables t and S A sample set of data that has been fitted in this way can be found in the Examples data subdirectory and is named Multiple rate 223 GraFit User s Guide 224 Advantages of Multiple Regression Use of multiple regression simplifies the analysis as the curve fitting need only be carried out once In the above example the value of A is fixed for the entire experiment and only found once for the multidimensional equation However if the data are analyzed by several applications of the first equation the A value would be determined several times Disadvantages of Multiple Regression The disadvantages of multiple regression are as follows e Conceptu
96. The contents of a GraFit workspace can be embedded or linked into other applications What is displayed is the contents of a particular graph window although the entire workspace information is copied To embed a GraFit graph into another application first activate the graph to use then choose the Select All command from the Home tab to ensure that all graph items are selected Note Itis extremely important to use the Select All command as no OLE information is copied unless all items are selected When all objects are selected the Copy command is replaced by Copy All Next choose the Copy All command Insert the information into an OLE compliant application using Edit gt Paste or Edit Paste Special as appropriate to the application in question 7 gt The Edit Insert New Object command in legacy mode The Edit Reset Original Size command in legacy mode The Edit Select All command in legacy mode The Edit Copy All command in legacy mode 76 77 215 GraFit User s Guide 216 CHAPTER 19 Data Fitting Basics This chapter describes some of the basic principles behind data fitting A more complete description of the theory is given in Chapter 20 and additional information can be found in several reviews Within GraFit data are fitted using the Non linear Fit or Linear Fit item windows which are described in Chapters 6 and 8 respectively When Is Data Fitting Appropriate Data fitting is the mathem
97. UAAIME Grabar n E E T 35 RIDDON OF Menand TOODI iii TEO 35 Ofhice 2007 SWVIERDHOM Ba senmenn AOE 35 Legacy style Menu and Toolbars orriren snn riaan EEE 36 Swapping Between Ribbons and Menu ToolbarS ooooonnccncnccccncnanincnnnnnononanos 37 TNO Galil WOKS Da Emna anaa A 38 Oro FIt KOMS sia 39 Mansen items ao 39 tem Representacion aci os 39 Editine tine Item NIMO cidad 40 Changing the Renie scsi 40 tem spece Oa oceyl aces oe eles heat vaca easter ea rea eata 40 Printing the Item Contents ccccsseccccssscccensccccescccseuseessuescessueceeseueeesseeseessueeessenseees 40 Pace SCCM tan 41 SEO IONNS odias 42 Grati Templates id ii dd aadezs 42 ACCESOS rt 42 WINAOW DISPIAY les ida 43 WY OF KING WIT FIGS ina Abiiibathivaes O 45 GA FIC WWORKSDACE Heston 45 OPEN al EXISTING Plena it 45 Opening a Recently Used HI ea a E 47 SAVING TEIG rosenen e a a Sateamedsesoeneeneeueaneees 48 Loading and Saving Individual GraFit Items ooccccccoccnnnnocnnnnononnnnanoncnnanonononos 49 Creating a NEW le a e a 49 IMPONE DATI Files Seea E aeaee 49 Grakit Template Fles eseriak ana A T AENEA AREN 49 Template DIreCtOr e Soen a a E E T 50 Data Tapies ra A ee AA 53 The Data Table MM iaa 53 Data IDE ido a aa T aa 55 Ignoring Individual Data Values occccooccnccnocnnonononcncncnnnnnanonononcnnnnanonnnnaninonnnos 56 Moving the Data Entry POSO oe 57 Selecting Regions of the Data Table ina 58 Selecting Part Of the Data Table cintia ri
98. Y Graph Wizard Set Initial Scaling Y axis Type Linear ha Autoscale Manual scaling 1 The Set Initial Scaling page allows the scale of the x and y axes to be specified By default the scale is set automatically and linear axes are chosen These can be altered as required it is also possible to adjust the scale settings later The text used for the x and y axis can also be entered 159 GraFit User s Guide Select Tick Styles XY Graph Wizard Select Tick Styles Daina oligo Set the initial tick styles for the graph The tick styles can be modified after the graph is created The Select Tick Styles page allows rapid selection of some of the more common axis tick styles More extensive tick style choices can be made once the graph has been created 160 Chapter 14 X Y Scatter Graphs Select Data Point Style not equation items XY Graph Wizard Select Data Point Style ax Style Appearance JOOWGG we 0 E CM Fill Mask curves Size Medium The Select Data Point Style page allows the data point representation to be chosen Size Data points are scaled relative to the overall graph width and so the absolute size will depend upon the graph dimensions Choose a size that represents your data appropriately with more data you should use smaller data points If very many points are present it can be more effective to set the Style as None and to join the data points with lines
99. Zoom Slider on the Status Bar It is also possible to adjust the magnification of the data table text using the zoom slider found on the status bar The zoom slider on the status bar Select the data table then drag the slider to adjust the magnification The magnification can be set more precisely by clicking on the zoom level text Grid Magnification Set the Grid Magnification by clicking on the zoom level text The View Zoom command in legacy mode 65 GraFit User s Guide Importing and Exporting Data Importing Data To import data into a data table position the data entry position where you want the data values to start and select the Paste Paste From command on the Table tab Select the file that holds the values to import and press the Open button The contents of the file will be inserted into the data table Importing Data as a New Data Table It is also possible to import a file into the GraFit workspace as a new data table item To import a data file in this way use Import Data command on the Home tab Exporting Data To export data from a data table into a file that can be read by other programs select the range of values to be exported and use the Copy To command on the Table tab Windows Vista and Windows 7 When running GraFit on Windows Vista or Windows 7 the program uses the new Vista style Save dialog box as shown below Copy To File G k User Documents Gra
100. a Properties Data Points Join Points Fitted Curve Data Source Eror Bars Axis Association X error bars None solute Proportional gt Explicit Y error bars None Absolute Proportiomal 10 0 Explicit General This section defines the general types to use for the error bars including the width and color of the pen used to draw them The Style choice is only available after error bars are selected in one of the X error bars or Y error bars sections Style Effect lett Line The error bars are drawn as simple lines ES T Bar The ends of the bars are delimited with a T bar The XY type option is only available if error bars are present on both axes XY type Effect HH Cross Error bars are drawn for the x and y axes separately g Diagonal 1 A single error bar is drawn which extends diagonally to show the x and y error extents a Diagonal 2 A single error bar is drawn which extends diagonally to show the x and y error extents 181 GraFit User s Guide The Source data entry must be completed if Explicit errors are specified for either x or y errors Select the data table that is used for the explicit errors from the list given X error bars This section is used to specify the type of errors present on the x axis Error type None Absolute Proportional Explicit Effect No error bars are drawn This is the default setting The size o
101. ackground Uncompetitive Inhibition In this multidimensional equation rate is monitored versus substrate and inhibitor The equation for uncompetitive inhibition is Vaak Giy v K 269 GraFit User s Guide T Uncompetitive Lineweaver Burk This transformation displays the Lineweaver Burk plot T Uncompetitive Eadie Hofstee Use this to display the Eadie Hofstee plot Enzyme Kinetics 2 Allosteric kinetics Hill The Hill equation describes enzyme kinetics for an allosteric enzyme For equation is al BE Vinax S 7 Hill Curve v _ kn S 2 4 si The Hill coefficient n equals 1 if standard kinetic behavior is followed but differs significantly from 1 if the enzyme 0 en kinetics are cooperative i 2 eee i 6 T Allosteric kinetics Hill Linear plot This transformation is used to display the linearized Hill plot where v V nax v is plotted versus S The slope is S K Enzyme Kinetics This equation fits the basic Michaelis Menten enzyme kinetics equation Vinay S Km S In this equation the rate v varies with the concentration of substrate S depending upon the maximal rate V nay and the Michaelis constant K w T Enzyme Kinetics Eadie am Hofstee al Eadie Hofstee Plot This transformation draws the linear dl Eadie Hofstee plot for fitted enzyme z e kinetic data A Rate Substrate 270 Chapte
102. ada Sececeade avivabedavavasbeadens 58 Selecting Entire COMMING ia 59 Copyins and Pastine Data diia 59 Copying and Pasting Regions of the Data Table occccoocccccnncnnncnacononanonnnns 59 COPYING and Pasting COlUMINS ainda 59 REMOVING Data Aa al ca 61 Clearing a Region OF Dita ib 61 Deleting a Region of Data tii diia 62 Inserting Values into the Table recimo e E AE 62 SOUND 63 Data Table APpesrantennini aia id 63 Colors ana Pontiac ratio 63 Number ROM barricadas 63 VMagniicilONa iia a 65 importing a a Exporting Data nn aii ida 66 A A RARE 66 Importing Data as a New Data Table ooccccocccnnoconcnnnnncnnnnnnnnnnnnnonononnnnnnanoss 66 EXPO Me Dala eem a a N 66 A A E E A A E A 67 General COLUMN PE Na 69 Pata TV E a a E a E a T a a ET 69 COMMU COMBINATIONS A a A 71 A A 71 Dato rte ss 75 Calculatea Vales dais 78 Data Roces IN digas 82 LOCKING a Data COIN sia 87 Noninear FIG TRG I saci ic sbi ovens scnav cna secsvs a EA 89 Overview Of Non linear Fitting ooocccccoooccnnnnonanonnnnnnnncnnnnnnnncnnnonnnnnnnnnonaronnnononannnnnnos 89 Status Bar INI CAL ONS associ cas cic eutectic T E etme alse tenaay A eeeyouaes 89 SETHI ES RN 89 EQU Oi noia 90 WEIERING aiara ana E EE ne react aan eso aceon cet E 90 Data Table oa 90 Data EVO e t diciones 90 X Data COJINES 92 Data COMO ciao 93 ENORCO IMM tad 93 CONSTANTS Panes E E OTOT 93 Linking Values to Data Table Entri8sS ccoooccncccccnnonononnnnncnnono
103. age controls this to 103 The View Options command in legacy mode 325 GraFit User s Guide use hard coded equations ensure that the Enable hard coded equation definitions box is checked Toolbar E Use large toolbar buttons Commands Enable hard coded equation definitions Inserting a Hard coded Equation Item Hard coded equation items are added using the Add Hard coded Equation command The hard coded equation window itself contains several panes that are used to define the equation Each of the panes in this item window is discussed below 326 Appendix 1 Hard coded Equations General Pane The General pane is used to define the location of the hard coded equation library file and shows a description of the equation as provided by the library supplier A Hard coded Equation General X Variables Parameters Constants Fonts Indents Location of equation library Y C Users Robin AppDlata Roaming WErithacusiGraFit 6 My Hard coded Equat hal eshmatez Es mo o ls CUUeE OO QENE aeS AUA OL thal Eslida Es Equation description The General pane of the Hard coded Equation item Calculates single phase association profile from a non zero initial value Locating the Equation Library File Click the browse button to locate the equation definition library file Once loaded the full path of this file is shown and a description of the equation is displayed X Variables Pane The X Variables pane
104. ailable within the Excel file If you wished to use these fitted parameters perhaps to perform further data manipulations within Excel you would need to open GraFit select the values and paste them into Excel Once again if the data or the fit changed then you would need to repeat this operation to get the updated values The following sections describe two approaches to achieving dynamic data transfer between Excel and GraFit Transferring Data using the GraFit Fitter Excel Template 300 The Excel file GraFit Fitter makes connecting Excel and GraFit a simple process that requires no programming skills This file has all the wiring necessary to drive the data transfer even allowing complex analysis scenarios with multiple data sets and GraFit objects all you need to do is define where your data are located where these data should be transferred to and where you wish the results to be returned Using GraFit Fitter to Fit the Example File id Hi e Dat R e Deselor Tear e 5 t age Layout Formula Data Revie c pe ea A Calidei it la p General A Fins i Ay A amp 7i AN rar ea Sort amp Find amp i Z7 mter Select BIU ANV ESIH Bus JA Oelete gt RERE 2a gt fede 1 Open the GraFit Fitter file this file is pre installed into your GraFit Data Sample Data directory Ses a Substrate Parameter Value Std Error Vmax 8 2788
105. ain GraFit window data tables graphs etc The contents of the workspace are stored together in a GraFit workspace file which has the GFF extension In addition to the contents of the workspace the current window sizes and positions are also stored in the workspace file Opening an Existing File Choose the File gt Open command to open an existing GraFit workspace file This will bring up the Open dialog box Windows Vista When running GraFit on Windows Vista the program uses the Vista style Open dialog box as shown below Open QU E t User Documents GraFit Data Se My Organize Gee Views fy New Folder Mame Date taken El Sample Data Favorite Links E Documents El GraFit Templates GraFit Data E Pictures Recently Changed Documentation on Enthacusnew Erith on Erithacusnew J Computer Desktop 3 Recent Places More Folders File name 45 GraFit User s Guide 46 The locations of your GraFit Data and GraFit Templates directories are added to your Favorite Links entries allowing you to navigate quickly to these The locations of these special directories are chosen from the Options command on the View tab Windows 7 With Windows 7 the Open dialog box includes the shortcuts to your GraFit Data and GraFit Templates directories under a GraFit 7 0 entry The locations of these special directories are chosen from the Options command on the View tab Op
106. al It is often difficult to visualize the problem in more than two dimensions e Finding initial estimates This is sometimes difficult for the same reasons e Difficulty of display The current version of GraFit does not support three dimensional plotting the display of four dimensional data is even more problematic Defining Equations Definition of equations having multiple independent variables is achieved in the same way as the definition of normal two dimensional x y equations but the equations have more that one x variable Fitting Data by Multiple Non linear Regression For a normal data set that contains x y data these data must be present in the data table with the x data and the y data in separate columns When multiple independent variables are involved there are special considerations as to how the data should be arranged Case 1 The equation has only two independent variables In many circumstances there will be just two independent variables in an equation In these circumstances it is common to collect one set of x y data at a particular x value then the next set of x y data at a different x value etc In the case of the example described above this would entail collecting A versus t at one particular S then another set of A versus t at the next S and so on When this arrangement of data is present it is convenient to enter the x y data into the data table and to associate the x value with the y da
107. and Pasting Columns Entire columns of data can also be copied and pasted Pasting into the data table when a column is selected results in the pasted values being placed in a new column in front of the selected column 2 The Edit Copy and Edit Paste commands in legacy mode 2 The Edit gt Copy command in legacy mode 59 GraFit User s Guide 60 1 Select the column s of data that you wish to copy Ola oo mila wo po 2 Select the Copy command 3 Select the column in front of which the data are to be inserted Data Substrate L ol oo on wo po 4 Choose the Paste command When pasting into a fully selected column you have the option to overwrite the selected data or to insert the new data in front of the selection the overwrite option is only available if the number of columns available to paste is the same size as in the selected region Paste Columns Insert data as new columns 5 Overwrite selected columns Chapter 5 Data Tables The data are then pasted into the table Substrate Ola oo on co po Removing Data Clearing a Region of Data The Clear command on the Home tab or the DEL key can be used to clear the currently selected data or columns 1 Select the data value s or columns or rows to be cleared Data Substrate L E J 4 z E j O a 24 The Edit Clear command in legacy mode 61
108. and also presents the results of the data fitting The item is created from the main GraFit window using the Add gt Non linear Fit command The non linear fit window itself contains a tabbed dialog box with several panes that are used to specify how the fit is calculated Each of these panes is discussed below Data fitting involves making appropriate selections in the Settings pane and possibly one or more of the other panes as described below then selecting the Fit Data command on the Home tab After fitting the data the results can be viewed from the Results pane Differences from Linear Fitting Non linear regression allows much more general equations to be used for analyzing data The biggest practical difference however is that non linear regression requires starting estimates of the unknown parameter values linear regression needs no such estimates Status Bar Indicators When a fitting item is selected the status bar shows the current fitting status indicating whether the item is Fitted or Not Fitted In addition the status bar shows when a calculation is in progress Whenever any calculation is being processed the calculation pane shows an animated abacus Fitting status pane Calculation pane Settings Pane The Settings pane is used to specify the main details of the fit 38 The Calculate gt Fit Data command in legacy mode 89 GraFit User s Guide 90 Ea Non linear Fit Fitting Criteria Results Number
109. aning Small capitals indicate keys to be pressed For instance A indicates press the A key If more than one key needs to be pressed this is written as for instance SHIFT END which would mean hold down the Shift key and then press the End key Monospace type indicates text that should be entered on the computer keyboard For example to obtain a directory of drive A when running a Command Prompt window you would enter DIR A RETURN Bold text is used for menu item commands e g the Edit menu Where cascading menus are present an arrowhead is used to separate the sub menus For example the Windows Control Panel application is started from the desktop Start Control Panel command Note Important information that should be noted is written in italics and separated from other text by horizontal rules as here 14 Chapter 1 Introduction Contacting Erithacus Software Technical Support At Erithacus Software we are working hard to provide you with intuitive technical products However if you find yourself with a technical question that you cannot resolve please contact our technical support department Registered users can obtain technical support from one of the following sources e Fax 44 0 1342 841 939 e Email support erithacus com e Internet http www erithacus com grafit Note Always quote your serial number and the product version which are shown on the About scree
110. ary OLE Automation tel Microsoft 12 0 dera i mee i y jasi Lig Ensure that the GraFit E 7 0 Type Li 7 Type Library is loaded Saeiionncel and selected if you VBAProject wish to access the seas ii mise GraFit automation AccessibilityCplAdmin 1 0 Type Library routines Arresso Fl FXnet Connect Anent 11 5 Tyne ihrary F ii h GraFit 7 0 Type Library Location DB ObjectFiles gfiWinBebug GraFit tb Language Standard The GraFit Type Library should have been loaded during installation If it is not present click the Browse button to select the GraFit tlb file that is present in the GraFit program files directory To use the GraFit automation routines it is necessary to make sure that this type library is checked as shown above It is necessary to select type libraries for each new VBA project that you create GraFit Automation Routines The following classes are defined by GraFit The main interface is the Graph interface which represents the embedded graph object itself To access the underlying fits and data the DataTable interface holds members controlling access to a particular data table the DataTables interface represents a collection of all the data tables in the GraFit object The FitResult interface holds members controlling access to a particular data fitting object the FitResults interface represents a collection of all the data fitting items within the GraFit object Class GraFit Graph GraFit Graph Membe
111. assays or Dose response curves 21 1C50 0 100 This equation fits inhibition data to a 2 parameter equation where the lower data limit is O and the upper data limit is 100 i e the data are both background and range corrected 100 a 1 72 In this equation sis a slope factor The equation assumes that y falls with increasing x T 1C50 0 100 show IC50 This transformation displays the IC value on the plot For best results set the curve resolution to High when using this transformation gt 1C50 background corrected This equation fits inhibition data to a 3 parameter equation where the lower data limit is O i e the data are background corrected _ Range poe 1 7 In this equation s is a slope factor The equation assumes that y falls with increasing x T 1C50 background corrected show IC50 This transformation displays the IC value on the plot For best results set the curve resolution to High when using this transformation T C50 background corrected show limit This transformation displays the limit value on the plot 273 GraFit User s Guide 2 1C50 full 4 param M Fits data to a full 4 parameter equation a E which is usually the best way to fit g s07 o oe results from RIA analysis or inhibition a 40 4 data see Halfman 1981 The binding Foy or inhibition is then characterized in ae ae AE Mi terms of an lC value 10 107 10 105 104 Ran
112. at File TIFF compressed or uncompressed For most purposes the compressed TIFF format is the most suitable Windows Vista or Windows 7 When running GraFit on Windows Vista or Windows 7 or later the program uses the new style Save dialog box as shown below The File Export Graph command in legacy mode Chapter 13 Graph Item 6 Export Graph As Oo E User Documents File narne Save as type TIFF file TIF E Export selection only a rt all Export a W Compress Browse Folders Windows 2000 or Windows XP Earlier versions of Windows use the older style File Save As dialog box Export Graph As Save in My Documents E GraFit Data My GraFit Data Amy Music my Pictures my Videos My Documents Grafit Data E Filename My Pictures Save as type TIFF file TIF ade O Export selection only Export all DPI 1600 Compress 155 GraFit User s Guide 156 CHAPTER 14 X Y Scatter Graphs The XY Graph Wizard Chapter 12 described the basics of creating graphs Selection of an X Y Scatter Graph produces the XY Graph Wizard to assist with the procedure all settings can be adjusted later if required Select Data Source Page Data set 4 Data All fits Ctrl click for multiple data sets Error column A Transform as You can use the Graph Add Data command to add more data sets to the plot The Select Data Source page allow
113. ata selected on the Settings pane is shown The graph is automatically scaled by default although the scale can be altered if desired To see the curve generated by a particular set of parameter values type in values into the Estimates value column of the grid When you move the data entry position the graph will be updated to show the curve generated by the current set of values These estimates should be such that the calculated curve falls close to the data values Just how accurate these parameter estimates need to be depends upon the equation in use and the data present However it is usually only necessary to produce a curve that roughly follows the data as shown above for the subsequent data fitting to find the best fit values 95 GraFit User s Guide When acceptable parameters have been found pressing OK will return these values into the Start column of the Estimatrix pane Fitting Criteria Pane As described in Chapter 20 non linear regression calculates best fit parameters by a series of iterations that produce successively more accurate results It is up to the program to decide at what point the values are deemed acceptable and at which point the calculations stop The criteria used to decide this are set from the Fitting Criteria pane It is likely that for most situations you will not need to change the default values on this page Ea Non linear Fit Settings Constants Estimatrix Fitting Criteria Results NumberFormat
114. ata and pass the results back to Excel turn to the GraFit worksheet page and click the Fit Data button 301 GraFit User s Guide E eis Embedded Graph in GraF Filter Microsoft Excel age layout Formula Dats R per Team 7 t o General A amet E Ay A EA B gt oee qe Ryes Sot Fiada Bs EE de Fike Seeds Mun or r rr 4 TJ Format gt o E F G H i Substrate N N N e Parameter Value Std Error Vmax 8 2788 0 5143 Km 2 0694 0 3724 HEHE Region for Fit invalidate Data A1 B31 Mee Me te iu z Embedded Graph in GreFa Fitter Microsoft Excel General tony Substrate Parameter Value Std Error Vmax 8 2788 05143 lt 0 3724 GraFit Fitter Reference The data are automatically transferred to the embedded GraFit analysis fitted then the fitted values passed back to Excel As a final refinement it is possible to specify that when the underlying data change in Excel the embedded GraFit analysis should be marked as out of date To do this on the GF settings page specify one or more data regions where changes are monitored In this case on worksheet Data the region where the raw data are located is A1 B31 Any changes to the raw data result in the embedded GraFit analysis being drawn with a dotted red outline and red background to show that it is out of date To upd
115. ata entry from column col and row row is returned The number formatting of the data value is as used by the source column If the data value at column col and row row is not zero then returns the text text If the data value at column col and row row is zero then returns the text text Description Returns the name of the x data column For data with a single dependent variable no is the fit number for data with two independent variables no is the data set number for data with multiple independent variables then no is the x data number Returns the name of the y data column For data with a single dependent variable no is the fit number for data with two independent variables no is the data set number Returns the name of the error data column For data with a single dependent variable no is the fit number for data with two independent variables no is the data set number Result Item Command per eS Lec paramname SIRO Example Parameter s LO paramno fitno paramno ELTAC paramno Chapter 13 Graph Item Description Returns the value of a fitted parameter from the data fitting The number formatting is taken from the result item format specification fitno is the fitting number 1 if there is a single fit paramno is the number of the parameter 1 for the first parameter 2 for the second
116. ate Sizing The Axis extent settings on the page shown above control the proportion of the graph over which the current axis extends When using multiple axes to show data of with different numerical ranges it is normal to have the axis set to occupy the Entire graph size However in some circumstances it can be useful to limit the axis to a small region For example this can be a good way to overlay residuals as illustrated below 195 GraFit User s Guide 196 Residual Rate O O 2 4 6 8 10 12 14 16 Substrate In this graph there are two y axis scales the first which holds the normal fitted data and the second that is used to plot the residual error The first y scale is set to occupy from O to 70 of the graph size the second is set from 70 to 100 Setting the Split Position Split axes are created by using axis extents less than the full graph size and by including a suitable Axis break symbol In order to modify the split position along the axis it is necessary to change the Start and or End position of the Axis extent This must be done for each axis concerned Tick Settings Each axis has full control over where and how often ticks are drawn For this reason when a new axis is created it often the case that the initial tick settings overlap existing settings from existing axes If this is the case it is necessary to modify the tick settings for one or more axis in order to obtain a suitable appearance
117. ate the analysis turn to the GraFit worksheet page and click the Fit Data button again The GraFit Fitter file is installed into your GraFit DatalSample Data directory The file is saved as an Excel Template which means that double clicking on the file icon will open the information into a new unnamed file Chapter 24 Passing Data Between Applications The file contains 4 worksheet pages The first Sheet 1 is blank and can be used to enter your data for analysis You can also add any number of additional worksheets as required ol 32 gt gt Sheet dE Gr settings 4GRABOUED A Worksheets in the GraFit Fitter file Ready 7 Setting up Your Data Analysis The first task is to set up this file so that it holds the data to be analyzed These data can be in any required format and may be included on the blank Sheet 1 worksheet page or on any additional page However do not alter the three worksheet pages that are specific to the GraFit Fitter file we will see how these are used shortly Ensure that Suitably Arranged Data are Available You can use Excel to perform any desired rearrangement of data but it is essential to ensure that in some defined part of the file there exists a region of cells that holds the data that will be passed to GraFit This needs to be a contiguous region where the data is present in vertical columns with the top row being used to generate the nam
118. ated when the source data change allowing powerful processing of the underlying data Example setting up a difference calculation As an illustration we will show how to set up the following data table in which the third data column holds the difference between the values in the first and second columns 67 GraFit User s Guide 68 Data 2 ABa falue 1 alue 2 alue 3 _ 4 3000 2 1000 3 5000 4 6000 6 2000 5 6000 Type area of 4 6000 3 6000 column 3 4 0000 3 9000 a m on amp oo BG The column type of the third column needs to be set by moving the data entry position to somewhere within this column and choosing the Column Type gt Column Combinations A B command from the Table tab Alternatively the Column Type submenu can be accessed by double clicking in the type area of the column For this column type setting the following dialog is displayed Calculate A B Data column A Value 1 Value 1 Value 2 Difference Difference Choosing Value 1 as the first column and Value 2 as the second gives the result above The values that are displayed in this column cannot be overwritten or edited and are updated whenever the values in the source columns changes The text color of the calculated values can be adjusted using the Preferences command on the Table tab but is typically shown a different color than the editable text in order to distinguish it The dialog settings for the calc
119. atical analysis of a data set in order to analyze trends in the data values This will usually involve linear or non linear regression analysis of these data values in order to define a set of parameter values that best characterize the relationship between the data points and an underlying theoretical model While many types of data can be usefully presented in graphical form not all such data are suitable for data fitting Types of Data In general data can be divided into three groups 1 No Systematic Trend There are many examples of such data and two are displayed below Cleland W W 1967 Adv Enzymol 29 1 32 Bevington P R 1969 Data Reduction and Error Analysis for the Physical Sciences McGraw Hill Dugegleby R G 1981 Analytical Biochemistry 110 9 18 Dress W H Teukolsky S A Vetterling W T amp Flannery B P 1992 Numerical Recipes in C Cambridge University Press Motulsky H J amp Ransnas L A 1987 FASEB J 1 365 374 Leatherbarrow R J 1990 TIBS 15 455 458 217 GraFit User s Guide 218 uN ab E 5 A rainfall chart Jan Febmar AprMayJun Jul AugSepOctNovDec Month 4 D gt E An elution profile from a purification column 0 0 5 10 15 20 25 Fraction Number Such data are typically presented as bar or column charts or as scatter plots with the data points joined by lines or possibly by a smooth curve As no systematic relationship describes the data they a
120. ation load it using the Add Stock Item command then click on the graph and select the Add Data command from the Graph tab The data source is the Non linear fit item and the Transform As box should be checked to allow the pKa Single Show pKa transformation to be selected Checking the Weighting 242 It is often useful when performing an experiment for the first time to estimate the type of weighting that should be used It must be stressed that for these checks to be meaningful a large number of data points should be available and the equation describing the data must be well established As was described in Chapter 20 plots of residual error Ay versus y or Ay y versus y give characteristic patterns for the common simple and proportional error distributions We will produce these plots for the data that have been fitted above Drawing Residual Plots We will plot the residual plot Ay versus y To make this plot we need a column of data containing the Ay values This can be created after data fitting by selecting the data table The Graph Add Data command in legacy mode Chapter 21 Worked Data Fitting Examples then setting the Column Type Table tab of one of the columns to Calculated Values gt Difference Difference Between Experiment and Fitted y Data Select the Non linear fit that was generated above and click OK It is advisable to set the column name so that you can identify it in the curren
121. ations A B Calculate A B pH Chemical Shift Differences Differences Relative Relative Using Robust Weighting Robust weighting is additional weighting that is applied to a data set It acts to reject data points that are very much in error and so removes the occasional rogue data point or outlier that may be present in a data set A complete description of the background to robust weighting can be found in Chapter 20 To show the detrimental effects of outliers on curve fitting and see how robust weighting eliminates these effects an analysis has been created which can be found in the file Robust weighting The data being have a linear relationship but are fitted using a linear equation defined as a non linear equation in order to make use of the robust weighting option that is available for non linear fitting Note Itis not possible to use a linear fitting item when using robust weighting The graph obtained by plotting the data with the best fit lines obtained with and without robust weighting is shown below 245 GraFit User s Guide 18 Data point rejected by 16 Robust Robust 14 weighting on weighting Robust 12 weighting off S 10 Q a 8 6 4 2 0 2 4 6 8 10 x Data As is apparent without robust weighting selected the presence of the outlier biases the fit considerably This is because the large sum of squares residual from this point has a disproportionate effect on the regression analy
122. b GraFit maintains a list of your most recently used documents To see this list open the File menu by clicking on the GraFit logo button GraFit logo button 47 GraFit User s Guide 48 Select a document from the list to re open it Pinning files to the list The list of recent documents shows files that have been most recently opened by the program If you wish to ensure that a particular document is kept on the list so that it will always be available you can pin any file to the list by clicking on the push pin icon iz Push pin icon un pinned Push pin icon pinned Click on the icon to toggle between un pinned and pinned Saving a File GraFit workspace files are saved using the File Save or File Save As commands The contents of the workspace are saved along with the current window positions and order When the file is reloaded these positions and order are restored Saving a File to Use With Older Versions of GraFit Newer versions of GraFit often have features that are lacking in earlier versions The latest versions of the program will read all earlier data files but older versions of the program may not be able to open files saved by the latest release To save a file using a format that can be read by older versions of GraFit version 5 and 6 use the File gt Save As command and set the Save as type to GraFit Workspace File versions 5 6 as shown below Save As gt cares NE
123. become reassociated with the default first axis Double dick on the data symbol to modify the associations lt Back Fish Cancel Choose the axis to remove then click the Finish button to delete it from the graph Any data or curves that are associated with the deleted axis become re associated with the default first axis You can modify axis associations by editing the properties associated with the data 193 GraFit User s Guide set To do so select the data symbol found in the data legend and choose the Properties command from the Graph tab or double click on the data symbol Working with Multiple Axes Axis Text and Axis Scale Text Each axis has a separate text legend that can be positioned and edited as required and a separate scale text that can also be formatted as required Data curve properties Absorbance Acetonitrile 4 6 8 10 42 y axis text 2 Fraction Number y axis scale 1 y axis scale 2 y axis text 1 Visibility of Axis Text and Axis Scale Text There are often cases where it is not necessary to show a separate axis text and or axis scale text for each axis The various items can be hidden by selecting the item then choosing the Hide Object command on the Graph tab Objects can also be hidden and unhidden from the General page of the graph properties double click on the main graph area or choose the Properties command when the main graph is selected
124. below PE 4 GraFit item open The icon is drawn normally Linear Fit open hi GraFit item closed The icon is drawn as a ghosted image which is lighter Linear Fit than normal closed Found on the Edit menu in legacy mode The Add menu in legacy mode The Edit Clear command in legacy mode 39 GraFit User s Guide Embedded object open for editing The object icon is shown overdrawn with hatched lines item open d Embedded object closed The icon is drawn ghosted Embedded item closed d l Linked object closed A shortcut overlay l21 is added to the icon Linked item closed Switch between item windows to edit their contents Information about using each of the GraFit item types is found in Chapters 5 13 Editing the Item Name Items are referred to by the name shown in the project window so it is useful to change this from the default to something more meaningful To edit the item name select the icon in the project window choose the Edit Rename command and type in the new name Item names can contain any characters but must be different from any other item name in the workspace Changing the Item Icon The icon that represents an item may also be changed This can be useful to allow similar items to be visually distinguished To change the icon select it and choose the Icon command on the View tab of the ribbon For each item type there are a number of alternative images that can
125. caling power setting allows a scaling factor to be applied to the numbers Before display the scale numbers are multiplied by 10 raised to the power of the value entered The power must be a whole number and may be positive or negative For example an entry of 4 would cause the value of 40 000 to be displayed as 4 An entry of 3 would cause the value 0 002 to be displayed as 2 0 002 x 10 2 A scaling power of O results in the actual value being displayed 10 1 Scaling power entries are very useful when dealing with large or small data values By scaling the displayed numbers it is possible to produce graphs that are correctly scaled but to enter conventionally ranged values into the data table For example time values in nanoseconds could be entered into the data table as whole numbers of nanoseconds then a Scaling power of 9 applied Axis Text Regions Axis text properties are controlled in exactly the same way as normal text objects See Chapter 13 for further information Legend Box Region 174 The legend box lists all the data sets currently present within the graph Legend Page Legend Box Properties Layout Number of columns A Custom margins and spacing Margins Order Spacing Chapter 14 X Y Scatter Graphs The current arrangement of items within the legend box is displayed This order can be changed by selecting an entry and clicking the Move Up or Move Down buttons Layout This section co
126. city of the first binding site T 2 Site Show Curve 1 This transformation shows the binding curve for the first binding site in isolation T 2 Site Show Curve 2 This transformation shows the binding curve for the second binding site in isolation Chapter 22 Equations Provided 2 2 Site with background This equation describes the situation where two independent binding sites are present but the y value at zero ligand concentration is not zero The equation for this is _ L Capay L Cape Ka L Kacy L T 2 Site with background Show background This transformation shows the background binding T 2 Site with background Show Capacity 1 This transformation shows the capacity of the first binding site This value is on top of the background T 2 Site with background Show Capacity total This transformation shows the total binding capacity for ligand on top of the background value T 2 Site with background Show Curve 1 This transformation shows the binding curve for the first binding site in isolation T 2 Site with background Show Curve 2 This transformation shows the binding curve for the second binding site in isolation 2 Adair 2 sites The Adair equation describes the binding of ligands to multiple sites on a receptor when the dissociation constants for binding of successive ligands are to be individually defined from the data The equation for n sites is n 1 L
127. ct the Edit Paste Special command 213 GraFit User s Guide Embedding and Linking without using the Clipboard Data objects can also be inserted into a target application directly using an Insert Object command which is often found on the Edit menu Insert Object Object Type Create New Microsoft Office PowerPoint Macro El Microsoft Office PowerPoint Macro El as Microsoft Office PowerPoint Presenta SEE Microsoft Office PuwePork Side Microsoft Office Word 97 2003 Doc Microsoft Office Word Document Microsoft Office Word Macro Enablec Organization Chart Addn for Microso Result Inserts a new Microsoft Office Word Document object into your document The Insert Object command allows the insertion of OLE objects into a document When the Create New button is selected an empty object of the selected type is inserted as a new embedded OLE item If the Create from File button is chosen it is possible to initialize the embedded item using the contents of an existing file or to create a linked object The Create from File option allows Create New File creation of an embedded object based Create from File WiErthacusnew documentation Manual on the contents of an existing file If in a F Link f addition the Link button is checked a aTi ah Ur wa ee linked object will be created rather than an embedded one Embedding and Linking with GraFit GraFit can act as both a container of OLE item
128. d If you wish to edit these files it is first necessary to unlock them which can be done using the Format Unprotect Sheet command By Fill ga Clear Cell Size yd Use the Format Unprotect Sheet command to unlock the jaan sample Excel files and allow them to be customized Re sates d In Excel 2003 the equivalent command is Tools gt Protection gt Organize Sheets Unprotect Sheet Rename 5heet Move or Copy Sheet Tab Color gt GraFit Automation Reference Advanced Using OLE Automation to control GraFit involves making calls to GraFit functions and subroutines that are exposed to programs that are automation enabled Not all programs are able to carry out automation and the examples shown in this manual use Microsoft Excel Using GraFit Automation with Microsoft Excel To ensure that you are able to access the GraFit programming interface it is necessary to include the GraFit Type Library GraFit tlb which is copied to the GraFit program directory during installation of the program Use of this requires the following preliminary steps 1 Ensure that the Developer Tab is Visible Excel 2007 Excel version 2007 does not by default show the programming tools Developer tab To reveal this first select Excel Options from the Office button 315 GraFit User s Guide Formulas Proofing Top options for working with Excel Save Show Mini Toolbar on selection Advanced
129. dd your own personal digital signature See http office microsoft com en us excel HA012317811033 aspx for more information Example Passing Data to and from Microsoft Excel One of the standard GraFit templates is the Enzyme Kinetics template see Chapter 23 Let us assume that we want to take data from Microsoft Excel and fit these data using this GraFit template The easiest way of doing this is to carry out the following procedures assuming that the data are present in an Excel spreadsheet Chapter 24 Passing Data Between Applications 1 The data to be fitted are present in a Microsoft Excel file Enzyme Kinetics Data 2 These data are copied and pasted into the standard GraFit enzyme kinetics template 3 The GraFit graph is embedded back into the original Microsoft Excel file 0 0 02040608 1 1 Substrate Substrate Parameter Value Std Error Vmax 8 2788 0 5143 Km 2 0694 0 3724 While this procedure produces the correct fit for the current set of data it suffers from a number of limitations 299 GraFit User s Guide e lf the data in the Excel spreadsheet change this is not reflected in the embedded graph To ensure that the graph reflects the current set of data these values would need to be copied and re pasted into GraFit e Although the embedded graph displays the fitted parameters the parameter values are not directly av
130. de 52 CHAPTER 5 Data Tables The Data Table Item To add a new empty data table to the GraFit workspace e Select the Add gt Data Table command The parts of the data table window are shown in the following illustration Column selection region Column resize region Type indicator row Column heading Data entry position Row selection region Splitter box Position row 1 column 1 in the above figure is marked by a dark outline this is the current data entry position Data entries are edited in situ by typing in the new entry When the entry is complete pressing RETURN TAB or a cursor key or clicking the mouse on a different position 53 GraFit User s Guide in the data table will accept the data and move the data entry position To edit the value at the current data entry position select the Edit Current Cell command or press F2 The following table describes the elements in a data table Certain sub regions are distinguished by the mouse cursor changing shape when it is over them Element Description Cursor Data table The data values themselves are listed in the data cy table Click the mouse on a value to move the data entry position drag the mouse to select larger regions of the data table Newly entered data is placed at the data entry position which can be moved using the cursor keys or by clicking the mouse on a different part of the data table Column selection region Click and drag
131. descriptive text The initial text used to describe the entry can be entered into this field Source Graphs are plotted using data that are present in one of the items found in the GraFit workspace The Source selection lists all items that contain suitable data for plotting and one of these must be selected Category axis Select which column holds the category names that are to be plotted Upper data The Upper Data selection is used to choose the data column that holds the values to be associated with the selected categories Error column Checking the Error column box allows a quick way of producing an error plot Select the column that holds the error values from the list More extensive error plotting options are available once the graph has been produced Lower data Check the Lower data box to generate a High Low chart where the entries have both an upper and lower value If this box is unchecked a standard chart will be produced if checked then it is necessary to choose the column that holds the lower data values for the High Low chart Lower error column When the Error column and Lower data boxes are both checked the error values for the lower data are selected from this box Chapter 16 Column and Bar Charts Set Initial Scaling Chart Wizard Set Initial Scaling Data axis text Text Values Autoscale Manual scaling Category axis text J Ted Categories The Set Initial Scaling page allows
132. ding This equation describes tight binding inhibition where I is comparable to E Note The concentration of enzyme is treated as a parameter rather than a constant This is because even in circumstances where you know the enzyme concentration it is unlikely that this will be known with sufficient precision After fitting your data to this equation always ensure that the fitted E value is sensible if it is not far higher or lower than the actual E then it is possible that you need to consider an alternative inhibition mechanism y y Elo lo Ka Y Edo llo Ka 4EJoKa Morrison J F 1969 Kinetics of the reversible inhibition of enzyme catalysed reactions by tight binding inhibitors Biochim Biophys Acta 185 269 286 Cha S 1975 Tight binding inhibitors Kinetic behavior Biochem Pharmacol 24 2177 2185 21 Tight binding offset This equation describes tight binding inhibition where I is comparable to E This equation assumes that inhibition does not proceed to 100 but that there is a background rate that is not being inhibited In real world situations this can happen for example due to residual impurities in the enzyme preparation If the background value is significant take care to question whether it is real See the description of the tight binding equation for more information Elo Mo Ka VCE lo Ka 4 EloKa v v background 215 0 b
133. e Axis Association parameters page described on page 183 Check the boxes of the axes with which your data should be associated In the figure shown above the graph has a single x and y axis and so no modification is needed 187 GraFit User s Guide 188 CHAPTER 15 Using Multiple Graph Axes When to Use Multiple Axis Scaling Graphs created using GraFit can optionally have several different x and or y axes which are independently scaled Overlaying Data that have Different Scales Multiple axes are useful when data need to be overlaid but have quite different numerical ranges For example in the plot below there are two separate y axes but a common x axis Two different data sets are plotted each associated with different y scales 6 Data associated O Data set 1 100 with 2nd y scale Dataset 2 a 80 E 4 Data associated D 60 w with 2nd y scale E E B 2 0 8 x lt AD 2nd y scale 0 0 1st y scale 2 4 6 8 10 12 Fraction Number XY scatter graphs can have as many different x or y axes as you require bar charts and column charts allow unlimited numeric axes the x and y axes respectively for these plots Split Axes Split axes are used when it is necessary to show some region s of a data set at a different scale to the remainder The example drawn below has a single split x axis GraFit uses multiple axes to construct the splits and permits any number of splits on both x and y axes 1
134. e Control Panel login screen Follow the instructions to create your account Once created you can then log in the your personal account Adding a Multi user License to your Administration Account When logged into you LCP account click Add New Software to add a multi user license to your account 26 Chapter 2 Installation and Activation x Add New software to your Account To add new software to your account you will need the Confirmation Key that was sent to you by email This email is sent to you after you activate your software for the first time Please Copy and Paste the Confirmation Key from the email here Ya 4 Enter Confirmation Key L Adding new software to your License Control Panel account You will have received the Confirmation Key in the initial email enter this and click Submit Once added you will be able to administer each multi user license that you register by clicking on the entry in the list Your Software Licenses single user software click on code to view details and manage Software CREE Activation EES 4 Activated Maximum Description Company Activation Code Date Activation email Computers Computers multiple seat software click on code to view details and manage Software Activation Activated Maximum Description Company Activation Code Date Activation email Seats Sents n s Erithacus ee GraFit Pro Academic Soin MA Wy 12 28 2009 r W erithacus com 0 10 Erithacus
135. e Style ax Fitting curve X limits check box for autolimit Siyle Miser fo Width O8pt E 2 End 10 Color i i Resolution Medium Fitting curve Choose the width line style and color of the curve that represents the fit X limits By default the fitted curve is drawn over the entire range of the x axis Some equations however are undefined over certain ranges for example at x lt 0 When the Start or End box is checked GraFit will set the limit automatically but by unchecking these boxes it is possible to define the range over which the curve should be displayed Resolution Fitted curves are calculated for a range of values between the Start and End limits The Resolution entry controls how many points are used in this range High resolution uses more points which takes longer and uses more memory In general use the medium resolution unless the curve has a sharp discontinuity e g when plotting a step function Under such circumstances use the high resolution setting 163 GraFit User s Guide Select Join Style not equation items XY Graph Wizard Select Join Style Join points Type None Type Chooses the type of join to use Effect Join Type None 164 Chapter 14 X Y Scatter Graphs Effect Join Type Line Bezier Three degrees of curvature are available slight medium and strong The illustration shows a medium curve Bezier curves give smooth lines between the p
136. e color of the grid lines and whether GraFit graphs show gridlines by default can be controlled from the Preferences command on the Graph tab The grid line spacing inches or centimeters depends on whether you are using the metric or the US measurement system this is set from the Options command Number Format page on the View tab The top and left margins are shown on the graph as solid rather than dashed lines the margin is specified using the File gt Print Page Setup command Element Outlines By default text elements and embedded objects are displayed with dotted and solid outlines respectively These do not show during print preview or printing and can be hidden by the Outlines command on the Graph tab which toggles their display Whether GraFit graphs show outlines by default can be controlled from the Preferences command on the Graph tab Adding Drawing Objects Various drawing objects can be added to the graph as controlled by the Draw command group on the Graph tab The View gt Grid Lines command in legacy mode The Graph gt Preferences command in legacy mode The View Options command in legacy mode The View gt Show Outlines command in legacy mode Draw menu or Drawing toolbar in legacy mode non wm uM O NO A Chapter 13 Graph Item Selection tool Round Rectangle Text abr Graph Wizard Ellipse Rectangle A p raw Polygon Selection Tool When the selection too
137. e if you are to have confidence in the calculated parameters High standard errors will be obtained if the data are of poor quality or if the data do not allow accurate estimation of one or more of the parameters Robust Weighting One of the assumptions of regression analysis is that all the data points have a normal error distribution However a common feature of actual experimental data is the presence of one or two observations that are well outside the error range of the other observations The stray data points are usually due to mistakes by the experimentalist incorrect addition of reagents inaccurate keying in of data etc and are termed outliers If these points are included in the analysis they will disproportionately affect the results since the square of the residuals for these points will dominate the fit It is possible to edit the data manually to remove such points but it is preferable to eliminate them in a less subjective manner Robust methods achieve this by applying an additional weighting factor varying for example from 1 0 for points that have low residuals to O for extreme outliers effectively eliminating such points GraFit employs optional robust weighting using the algorithm of Mosteller amp Tukey as implemented by Duggleby An additional bisquare weight b is incorporated into the analysis and is calculated as D u iflu lt 1 l 0 if lu gt 1 Here with z being the residual weight
138. e real experiment and it can be preferable to treat this background as a separate parameter to be obtained from the data fitting The equation is Bound Binding Curve Show Capacity Show K Show Background 275 GraFit User s Guide 276 O ld y K L ackgroun Here the parameters are the same as for the single site ligand binding except for the inclusion of a background or offset value T 1 Site with background Scatchard Use this transformation to display Scatchard plots for binding data containing a background value T 1 Site with background Show background This transformation shows the background value T 1 Site with background Show Capacity This transformation shows the capacity T 1 Site with background Show Kd This transformation shows the dissociation constant Ky For best results set the curve resolution to High when using this transformation 2 Site re Often there is more than one type of site involved in binding the ligand This 4 equation describes the case of two sites characterized by individual Ky and Capacity values The equation is L Capa L Capoz nn a Kaca L Kay L Bound Ligand Binding 2 Sites T 2 Site Show Capacity total This transformation shows the total binding capacity for ligand T 2 Site Show Capacity 1 This transformation shows the capa
139. e same for an entire set of x4 y substrate rate data pairs Under these circumstances it simplifies the data entry to associate a single x value with a whole column of different y values This is done by specifying the column type as y Data with Associated Value The y Data with Associated Value command is also available from the Table tab Y Data Column with Associated x n Value Associated values Column type label Label x2 2 Load Default Label Use the following placeholders where you wish to display the x n data value s 2 x 2 3 43 4 x4 5 f5 It is found on the Data toolbar in legacy mode Chapter 5 Data Tables The x data value is entered in the above dialog and is normally displayed in the column type area Data columns that have associated values are used when fitting to equations with two independent variables See Chapter 6 for information about using this option when fitting data Up to four values can be associated with a data column allowing Xx x3 X4 and xs values to be stored if required for equations having up to five independent x variables If your equation definition has only 2 independent variables the values for x3 X4 and x are ignored Column Combinations These column types allow the values within the designated column to be the result of an arithmetic combination of two other columns In each case the number of calculated entries depends upon t
140. ear equation that is analyzed by GraFit is of the form Chapter 19 Data Fitting Basics y f x where the right hand side of this expression may contain one or several parameters Such an equation has one dependent y and one independent variable x In addition GraFit can be used to analyze equations with more than one independent variable i e y f X1 X2 X3 a0 In this case the analysis is termed multiple non linear regression GraFit allows unlimited independent x variables as well as unlimited numbers of parameters Note however in all cases it is assumed that only the y axis data contain errors Note Multiple regression is sometimes also referred to as global analysis Uses for Multiple Regression Multiple regression is essential when the data to be examined are inherently multidimensional Examples include peak shape analysis of 2D spectra However there are several situations where multidimensional data are collected but are traditionally analyzed by a performing a series of normal two dimensional x y curve fitting Example The progress of an enzyme catalyzed reaction at a fixed concentration of substrate is given by the equation At Ao 1 ee Here A is the extent of reaction at time t A the maximum extent of reaction and k the rate constant at this concentration of substrate The value of k from this equation is related to the kinetic parameters by the equation Vax S K km
141. ed at any time An activation code can only be used to top up another code if the top up code has not already been activated and can only be used once for this purpose Note It is only possible to top up a multi user license using one of the same type For example a 10 user license can only be topped up with another 10 user license Please ensure that you purchase the correct license type when you wish to extend your license Frequently asked Questions About Activation and Licensing Single User Licenses What happens to my installation if my computer is damaged or replaced Where possible you should explicitly deactivate your GraFit installation on any old computer then reinstall on the new one However there are circumstances where this is not possible typically if your computer breaks or is stolen Under these circumstances you simply need to reinstall GraFit on the new computer using your activation code and password If you have forgotten your password then this can be resent to you by clicking the Forgotten Password button on the installation dialog box In the event that the new activation exceeds the permitted activation count you will still be able to proceed though you will also have to reactivate any GraFit installations on other computers see What happens if try to activate more copies than allowed by my license What happens if try to activate more copies than allowed by my license If you try to activate GraFi
142. ed by the a priori weight and N the number of observations Zi uU 6 Yl zi N Including robust weighting will result in improved estimates for the parameters in the presence of outliers This is however gained at the expense of more calculation which will slow down the fitting procedure a Bevington P R 1969 Data Reduction and Error Analysis for the Physical Sciences McGraw Hill Book Company Mosteller F amp Tukey J W 1977 Data Analysis and Regression Addison Wesley Reading MA USA pp 353 65 Duggleby R G 1981 Analytical Biochemistry 110 9 18 87 88 233 GraFit User s Guide The effect of using robust weighting is seen in the graph below Data point rejected by Robust Robust weighting on weighting Robust weighting off y Data 0 2 4 6 8 10 x Data The data which contain a single outlier have been fitted to a straight line with and without robust weighting enabled When robust weighting is omitted the presence of this outlier markedly affects the calculated results With robust weighting selected however the rogue data point is rejected and the calculated line now correctly follows the main trend Determining the Most Appropriate Weighting 234 For most experimental data the error is commonly either independent of the value of the observation simple or constant errors or is proportional to the y data value proportional errors Simple errors may be caused by instr
143. ed data The transformed curve definition uses the X name and Y name entries which correspond to x and y in the above equation The syntax used for this definition is as described for equations see Chapter 9 and can occupy more than one line if required 1 I y x Fonts and Indents Panes These panes control the way that the results are formatted The contents of these panes are common to several different item windows and are described in Chapter 7 Compiling the Transformation To compile the equation choose the Compile command from the Transform group on the Home tab Compilation translates your definition into an internal form that can be executed rapidly During compilation the definition is comprehensively checked to ensure that the correct syntax has been used If an error is detected a message box will be displayed informing you of the mistake after pressing the OK button to continue the cursor will be placed at the most likely place of error GraFit An Error Message Box To obtain further information on the specific error click the Help button or press the F1 key Once the transformation has been correctly compiled it can be used for graph plotting 129 GraFit User s Guide 130 CHAPTER 11 Notepad Item Uses of Notepad The notepad provides a text editor window where information can be stored in the GraFit workspace Uses include making notes about the data the analysis of the results and
144. ed there 19 GraFit User s Guide 20 Using the Evaluation Activation Code When you receive the email with your code paste this into the Activation Code box and click the Activate button During activation you will be asked to provide a password which will be needed in future if you reactivate or deactivate the software and an email address that is used to allow automatic reminders to be sent if you forget your password During the evaluation period the software is fully functional After the evaluation period has ended it will no longer be possible to run the software unless you purchase and enter an evaluation code GraFit Untitled Text Pa a 3 Import Data A 5 Ere 5 LJ NON EN TENNE a Add Stock Item tiles f ik mll Edit Text Add ES A E Paste Undo Redo G ay Store Stock Item Items Clipboard Edit y Evaluation mode You have 0 days of your evaluation period remaining Activate GraFit running in evaluation mode Purchasing a Full Activation Code Full Activation codes can be purchased online by following the link on the Activation dialog box There are two distinct types of full activation code single user activation codes and multi user activation codes More information about multi user codes can be found later in this Chapter Activating the Software Once you have purchased a full activation code paste this into the Activation Code box and click the Activate button If you purchased GraFit
145. en JCJ a gt Robin My Documents GraFit Data z Organize New folder gt G Grafit 7 0 A Mame Date modified Type E GraFit Templates az Sample Data 21 02 2009 00 20 Shortcut E GraFit Data r Favorites la Recently Changed Le Public Ga F drive Y Backup 7 Computer Desktop p Documentation ERITHACUS mM Downloads LY Erith ERITHACUSNEW pM ERITHACUSNEW ca Media Archive Y File name Windows 2000 and Windows XP Earlier versions of Windows use the older style file open dialog box 11 The View gt Options menu in legacy mode Desktop fa My Documents a My Network Places E GraFit Data E File name GraFit Templates Files of type 7 Open Ctri O Clase Save Ctrl 5 Al Save As Y Send Mail 2 Properties All GraFit Files Opening a Recently Used File Recent Documents test CPM kinetics 2009 07 22 Matrix_30f6 Standard curve Robust weighting PH Titration Paired linear regression Multiple rate Lo 164 1 Jin e lla Ira Multiple linear Hemoglobin saturation Enzyme Inhibition DNA hybridization Dixon plot Competitive Inhibition Chapter 4 Working with Files ERY YY Y Y Y Y Y Y Cancel The buttons marked GraFit Data and GraFit Templates allow you to rapidly to the default data and user template directories respectively The locations of these special directories are chosen from the Options command on the View ta
146. en permission of Erithacus Software Limited Erithacus Software Limited PO Box 274 Horley Surrey RH6 9YJ UK VERSION 7 Last revision January 2011 Trademarks GraFit is a tademark of Erithacus Software Limited Microsoft Windows NT are trademarks or registered trademarks of Microsoft Corporation Other brands and products are trademarks or registered trademarks of their respective holders and should be noted as such Citing GraFit in Publications When citing GraFit simply treat this software publication as you would a book For example Leatherbarrow R J 2010 GraFit Version 7 Erithacus Software Ltd Horley U K Contents Hodur caves tvees unt yansy ues yeusvoutpaceneernee duane aneummevereuantvateeeemeeerys 13 WEIMAR 13 key Features O POPE 13 ADOUE IS GUISA 14 Notationai CONVEN IONS earra icaio 14 Contacting Erithacus Software ccccccoonccnnnononnnnnnonanonnnnnnnacnnnnononcnnnnnnnncnnnnnnancnnnnnnncnnnnnos 15 Technical SUPDO aaa 15 A 15 Installation and ACTIVACION iiias isan emanate ness 17 System Requieren 17 Mie Gia lel PAC KASS xa sete a aces nies EA 17 AAA e O O ee 17 onime Instala Muente 17 CD ROM installato Musa 17 Instalacion AP ROCOSS tr 18 Activating tne Grati Mia 18 Activation Dial g BO aaa 18 Running in Evaluation Mode ccoooccnccnccnncnncnnncnncnnononennnnonononnnnnnnnonnnnnnonnnnnnanininnnos 19 Purchasing a Full Activation Code cccccooccnnccncnnccnonnnonononnnnanonononnnnnnanononnncn
147. en constants are included in an equation used for non linear regression their values are entered into the non linear fit item window as described in Chapter 6 Chapter 9 Equation Item Enzyme Kinetics General A Variables Parameters Constants Definition Estimates Fonts Indents Constants Description The Constants pane of the Equation item The organization of the Constants pane is very similar to that of the X Variables pane and the definition of Symbol and Description names is performed in an analogous manner see the X Variables Pane section above The number of rows of information that are entered into the grid defines the number of parameters in the equation If no constants are required as will be the case for many equations this page should be left blank Definition Pane The definition pane describes the functional form of the equation using the Symbol names of the x variables the parameters and constants if any entered as on the earlier panes 115 GraFit User s Guide 116 Enzyme Kinetics General X Variables Parameters Constants Definition Estimates Fonts Indents name k Equation definition 1 v Vmax S Km 5 The Definition pane of the Equation item Symbols double dick to insert into definition X variables Parameters Constants Vmax Em All equation definitions that are used within GraFit must have the functional form y f x i e it must be possible to describe the va
148. en no initial estimates were used these options are unavailable as in the figure above as the estimates were generated automatically for the equation used 81 GraFit User s Guide 82 Difference Use this column type to calculate the difference between the experimental and the calculated y values using the fitting model described in a selected workspace item Difference Between Experiment and Fitted y Data Fitting results Fit Single Fit number Data 1 2 3 A A yes _ diference Column Initial Rate Mane 0 0000 110 0000 1 3651 1 0000 60 0000 1 4794 2 0000 60 0000 1 1119 3 0000 45 0000 0 8357 4 0000 35 0000 0 6219 5 0000 28 0000 2 2154 1 oo oo om ta as _ 2 4 Plots of difference versus x or y values are very useful to indicate whether the calculation accurately reflects the variation in the data A good fit is characterized by a random distribution of small differences between the raw and fitted data Data Processing The column types under this submenu allow various types of processing to be performed to generate data values for the column Calculated Function The calculated function column type fills the selected column with the results of a user defined function This column type can be used to perform sophisticated processing of data including background subtraction calculation of logarithmic values and complex rescali
149. ends on how may entries are present in the analysis list In this example are 8 separate analyses 11 Results GraFit1 we 12 Results GraFit2 W 13 Results GraFit3 We 14 Results GraFit4 w 15 Results GraFit5 Wo 16 Results GraFit6 we 17 Results GraFit7 wf 18 Results GraFit8 We 19 an a we a ors A blank entry marks the end of the analysis list Data Section For each of the embedded GraFit graphs it is possible to transfer one or two regions of data from Excel into a named Data Item within GraFit The location of the source data within Excel is specified in terms of the worksheet page and region of cells that hold the information the coordinate of the top left and bottom right cells needs to be specified The data are transferred to GraFit and placed in the Data Item that is specified the data are transferred starting with the designated Start Column For example if the Start Column is 1 then the data are copied into the first column of the Data Item The top row of data within Excel has special meaning in that these data are used for the column names within GraFit see above 305 GraFit User s Guide Data 1 Destination Destination GraFit Page Top Left Bottom Data Start Right Item Column Working A 112 Data 19 Specifying the Data locations for the ae a Se a i e first data region on the GF settings 4 Working A41 151 Data 1 hi page gt Working A54 164 Data 1 Pi Working A67 177 Data 1
150. ensional array Dim c 1 To arr size As Variant Dim d 1 To arr size As Variant For i 1 To arr size Let c i b i 1 Let d 1 afi 1 Next Rem Pass the data to GraFit Call GF SetColumnData CalcSheet GFObject GFData Call GF SetColumnData CalcSheet GFObject GFData Rem Update the names of the columms within GraFit Call GF SetColummName CalcSheet GFObject GFData Worksheets CalcSheet Range Al Value Call GF SetColumnName CalcSheet GFObject GFData Worksheets CalcSheet Range Bi Value Rem Wait until calculation done Call GF WaitForCalculations CalcSheet GFObject End Sub a The FitData subroutine from the Linked data IC50 sample file Chapter 24 Passing Data Between Applications This subroutine gathers together data from Excel and then passes the information to GraFit using a set of GF_XXXXXX subroutine calls These subroutines are helper functions that make using GraFit automation easier and are collectively found in the sample data file in GF_module The VBA program code for these is present in the Sample Data directory in a file called GF_module bas Note If you are planning use the automation features we recommend that you edit the sample Excel files to see how this has been implemented Sample Data Files Installed When GraFit is installed some sample Excel data files are copied into your GraFit Sample Data directory These files illustrate some of the ways in wh
151. epresented by a different icon e Double clicking on a template file opens a copy of the file rather than the file itself e Templates stored in the user or group template directories can be rapidly accessed using the File gt New command Creating a Template File To store a workspace as a template file select the File gt Save As command and save as type GraFit Template File to create a template file that can be read by earlier versions of GraFit save as type GraFit Template File version 5 6 If you wish to access this template using the File gt New command ensure that the file is saved into your user or group template directory see the following section on Template Directories To help with this the Save As dialog box contains a button that will jump immediately to your user template directory Creating a File Based on a Template The File gt New command creates an untitled file based on a selected template This command in described in the following section Chapter 23 describes the standard templates that come pre defined with GraFit Template Directories There are two directories that have special significance when storing template files These are the user and group template directories the location of which is specified from the Options command on the View tab Directories page Directories Data Data Templates User C Users Robin App Data Roaming Enthacus GraFit Group CA ProgramDlata E
152. er is not appropriate for these data 262 Rate 1 Rate 1 Rate Competitive model 6000 4000 2000 Rate POT STI roo 0 008 0 006 0 004 0 002 6000 4000 Rate 2000 0 008 0 006 0 004 0 002 Substrate Reduced Chi 64387 8893 Parameter Value Std Error Vmax 5320 8514 211 8898 Km 0 2646 0 0423 Ki 0 1336 0 0203 1 Rate 1 Substrate Mixed type model Rate Substrate Reduced Chi 65843 5622 Parameter Value Sid Error Vmax 5379 6409 226 6466 Km 0 2752 0 0460 Ki 0 1512 0 0330 Ki value 13 3794 11 8828 PORFA REA 1 Rate 1 Substrate 6000 4000 2000 0 008 0 006 0 004 0 002 6000 4000 2000 0 008 0 006 0 004 0 002 Non Competitive model Substrate Reduced Chi 232468 9108 POT PTTR PR TES Parameter Value Std Error Vmax 6365 0292 519 7938 Km 0 5911 0 1403 Ki 1 1252 0 1826 1 Substrate Uncompetitive model Substrate Reduced Chi 415505 5294 POT PTTR PR TOS Parameter Value Std Error Vmax 6478 1123 819 7069 Km 0 7032 Ki 0 8107 1 Substrate Chapter 21 Worked Data Fitting Examples 263 GraFit User s Guide 264 CHAPTER 22 Equations Provided GraFit comes with a large number of
153. er of the current row This value is most useful in conjunction with the Data function rad Returns a random number between 0 and 1 grod Returns a random number that falls within a Gaussian distribution that has a zero mean and unit variance The following examples show how calculated functions might be specified and used Process Result field Background Data 1 Row 0 234 subtraction Log log Data 2 Row calculation Complex Data 1 Row 1 34 rescaling Multiple line If ValidData 1 Result Data Else SetBlank Ena E statement Data 2 Row Data 3 2 Description Subtracts the value 0 234 from the values in column 1 Takes the logarithm to base 10 of the values in column 2 For each row in column 1 multiplies this value by 1 34 then subtracts the data in column 2 which has been divided by the value in column 3 row 2 For each valid data entry in column 1 sets the corresponding entry in the current column to be 23 3 greater Note It is essential to inform the program by checking the appropriate boxes which columns if any the calculation depends upon Failure to do this will result in the calculation not being updated correctly when these values change Chapter 5 Data Tables Derivative The derivative column type fills the column with the calculated derivative order O to 5 of a selected source data column The contents of the source column are not affected by thi
154. es These panes control the way that the results are formatted The contents of these panes are common to several different item windows and are described in Chapter 7 100 CHAPTER 7 Formatting Item Windows Formatting Options Many GraFit Item windows are built from a tabbed dialog box that allows various settings to be entered The Linear Fit Non linear Fit Equation Hard coded Equation and Transformation definition items are all built in this way In each case the settings in the window can be displayed using the File Print Print Preview command and printed by the File Print command Each of these item windows includes a Fonts and an Indents pane that control the way the information is displayed when printing Fonts Pane The Fonts pane selects the fonts to use when displaying the contents of the item window Ea Non linear Fit Settings Constants Estimatrix Fitting Criteria Results NumberFormat Fonts Indents Item Normal text Font Font style Times New Roman Regular Traditional Arabic Itali 11 The Fon n he Non O Trebuchet MS Bolc i Selves PR nan O Tunga Bold Itali 14 linear Fit item O Tw Cen MT O Tw Cen MT Condensed Q Tw Cen MT Condensed Extra Bold 3 Verdana Script Western Y The File gt Print Preview command in legacy mode 101 GraFit User s Guide This pane behaves in the same way as a standard Windows font selection dialog The fonts to use can be set f
155. es of the columns when this is passed to GraFit The region can be any width and any height A B 1 Pe J 3 af 4 4 5f i ll Data values will be transferred between a Ca designated region on one of the Excel worksheet 9 pages This region spans cells A1 top left to B8 bottom right A Beer oe CO De oO 303 GraFit User s Guide 304 x E Data X fe Graph 3 and a named Data Item within the embedded GraFit analysis file ah o AS A A we Hint If you have a pre existing Excel file that you use to analyze data you can copy and paste the relevant worksheet pages into the GraFit Fitter file If your file does not already hold the data in an appropriate column format as shown above use the ability of Excel to produce a tabulated set of information on a separate worksheet page that is just used to hold the intermediate values See the example files for a case where this has been done Embed One or More GrarFit Files into the Excel File After creating a GraFit file that performs the data analysis you require this file is embedded within the GraFit Fitter Excel file To do this set up your GraFit file to perform the data fitting select all items in one of the graph window and use the Copy All command Paste the graph into Excel either on Sheet 1 or any other worksheet page Once pasted select the graph and give it a name by which to can refer to it within the Excel
156. et The x and y axis labels are taken fromthe headings on the Calc sheet If Auto Update is checked then the fitis automatically updated Inhibitor wheneverthe raw data values change if Parameter Value unchecked then the ee calculations are only Y Range 103 9187 updated when the Fit Ic 50 4 7552 Data Slope factor 3 7627 y i Databuttonis Background 3 2916 DI ee Bist 8 8 a b SDRE B eles eos SR E Boon a 311 GraFit User s Guide 312 Ntra if Detailed view of how the data are linked c together between the E TE A e mh 4 worksheets IF ISBLANK Raw Data 1B4 Raw Data B4 How is Data Transfer Done The data transfer is achieved using VBA To illustrate the main data fitting subroutine FitData in the sample above is reproduced below Linked data IC50 xlsm Modulel Code o Jasa General FitData Public Const arr size 30 Public Const output range F2 H5 Public mydata As Variant Public Const CalcSheet Calec Public Const GFObjectS GraFit 1 Public Const GFData Data Public Const GFFit S Non linear Fit pp il Public Sub FitData Rem Get the data from the worksheet Dim a As Variant Dim b As Variant a Worksheets Calc5heet Range A2 A Format 1 arr size Value b Worksheets CalcSheet Range B2 B Format 1 arr size Value Rem We need to put the data into a 1 dim
157. etc Returns the parameter name from the data fitting See the parameter description for more information Returns the error value from the data fitting See the parameter description for more information The following text would allow a data value present in the first row of the second column of table My Data to be included within the graph The graph text is dynamically updated whenever the underlying data value alters The answer to life the universe and everything is My Data Data 2 1 Answer 42 0000 Graph The answer to life the universe and everything is 42 0000 Entering Field Codes into Graph Text the Easy Way An easier way of entering field codes is to use the Field Code command on the Graph tab First create a text item on the graph then use the Field Code command where you wish the field code to be placed The following Text Field dialog box controls the entries 65 The Edit Field Code command in legacy mode 149 GraFit User s Guide Text Field Source tem Text Strings Command Data Column Number 2 Returns the data value from the O The Text Field dialog is used to create and edit field codes Row Number 1 within graph text Enzyme Kinetics Data Chose the Source item set the Command as required then enter suitable values for the parameters as prompted To edit an existing text field code position the cursor between the two curly brackets and enter the Field Code
158. eter If the first ionization results in increasing activity and the second destroys activity this results in a bell shaped curve The equation assumes that activity is zero at low and high pH T pKa Double Bell Curve 1 Bell Curve st Curve 2nd Curve This transformation is used to display the effect of the first ionization in the bell shaped curve Chapter 22 Equations Provided T pKa Double Bell Curve 2 This transformation is used to display the effect of the second ionization in the bell shaped curve T pKa Double Bell Limit This transformation shows the upper limit of the bell shaped activity profile 2 pKa Single This equation is used when the observed Ml eas parameter varies from one value at low pH lla UE to another at high pH with the variation dl Show Limit depending upon the ionization of a group 2 The pK value of this group determines the aa inflection point of the curve The equation 5 6 7 8 9 10 is Limit Limit 10 PKaq Limit 4 i 10 PH PKa m 4 1 In this equation Limit and Limit are the limits of the titration curve at low and high pH T pKa Single Linearized plot This transformation displays the line for the linearized titration curve T pKa Single Show Limit 1 This transformation displays the limiting y value at one extreme of pH T pKa Single Show Limit 2 This tra
159. expires soon Your multi user software license expires in 3 days time Multi user license expiry warning What Happens if my Multi user License Expires If a multi user license expires then the next time a user of code tries to run GraFit they will see the following dialog box 29 GraFit User s Guide 30 Grafit 7 Software Activation Activate GraFit 7 Car TS A Activation Code full or evaluation WI LLE EL Evaluate GraFit Y Your multi user software license has ase S of tware Activation i dialog box displayed when a multi user license This software was activated by a multi user license that has now expired has ex pir ed It is also possible that your computer cannot connect to the internet to walidate the license Try again with a valid internet connection or contact your license administrator to get your existing license renewed To reactivate the program using a different license enter a new Activation Code into the box above and click the Activate button Change Proxy Settings Purchase activation code online This dialog box is displayed either if the multi user license has expired To continue working with GraFit either renew the existing multi user license at which point all users of this license will then be able to use the software again or purchase a new single user or multi user license and reactivate the program using the new license Note Ifyour computer is not connected to the internet for a
160. f the GraFit object Y strGFFit Name of the fitting item within the GraFit object ee The parameter number to return start at 1 fitNo The index start at 1 of the fit in the object Chapter 24 Passing Data Between Applications Public Sub GF SecColumnData ByRet strCalconest As String ByRef strGFObject As String ByRef strGFData As String ByVal iColumn As Integer ByRef data As Variant Fills a column of data in an embedded GraFit object strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object strGFData Name of the data table item within the GraFit object Column The number of the data column start at 1 data The array of data start at 1 to be inserted Public Sub GF WaitForCalculations ByRet strCalcsheet As String ByRef strGFObject As String Waits until embedded GraFit object has finished calculating strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object Public Sub GF SetColumnName ByRef strCalcsheet As String ByRef strGFObject As String ByRef strGFData As String ByVal iColumn As Integer _ ByRef strColumnName As String Sets the name of a data colmn in an embedded GraFit object strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object strGFData Name of the data table item within the GraFit object Column The number of the data column start at 1
161. f the error bar is the same for each data point of the data set this size is entered into the field to the right of this button The size of the error bar depends upon the magnitude of the data point the size is expressed as a percentage of the data point value and is entered into the field to the right of this button The size of the error for each data point is specified individually The error values must be stored in a data table which is specified in the General section and the size of the positive and negative parts of the error bar is set separately The data column that holds the positive error values is selected from the list marked If only this option is chosen the error bar just has a single lobe u n To specify additionally the size of the negative lobe click the button and select the column that holds the value of this error If you select the same column as selected for the positive error this will result in symmetrical error bars The Invert check boxes allow the direction of the error bars to be reversed drawing the error bar in the opposite direction Some examples of error bar types are shown below No error bars 182 Absolute error bars on the y Proportional error bars on axis the y axis Chapter 14 X Y Scatter Graphs Explicit error bars on the y axis the positive and negative lobes each use the same error column Explicit error bars on the y axis only the positive lobe is shown
162. f using linear regression of a linearized function for analyzing data Chapter 9 Equation Item The Estimates Pane Enzyme Kinetics General X Variables Parameters Constants Definition Estimates Fonts Indents V Estimate from linear transform V Omit values equal to zero Transformed x data Transformed y data Definition f xdata vydata Definition f xdata ydata vdata xdata The Estimates pane of the Equation item Definition intercept gradient 1 gradient To specify that initial estimates are provided for an equation the Estimate from linear transform box should be checked If you do not want the program to calculate initial estimates simply leave the box unchecked Defining How to Rearrange the Data Points For our current equation the rearranged plot that we require is of v S against v i e y x versus y The X Data and Y Data entries are used to define the data points for such a plot note that the stored data points in the data table are of course unaffected For this equation the definitions are ydata and ydata xdata respectively The symbols xdata and ydata are predefined by the program to represent the x and y data points These definitions allow the program to perform the rearranged plot and any valid single line expression can be used The grid at the bottom of this page is used to define how we obtain our initial estimates from linear regression on the rearranged pl
163. familiar with the basic functioning of the program and if not you may like to consult the earlier chapters Using One of the Equations Provided We will fit the data obtained from an NMR titration curve to obtain the pK of an ionizable group Background The position of a peak in the NMR spectrum the chemical shift position resulting from a magnetic nucleus that is part of a titratable group depends upon the equilibrium between the two ionized forms For example in the following equilibrium A Ht AH Nuclei close to the ionizable group A will have chemical shift values that depend on the ratio of A and AH By definition the acid dissociation constant Ka is given by A7 H Ka Tam It is usually more convenient to use pK values where pK logioKa The variation of the ratio of A and AH is given by the Henderson Hasselbalch equation pH pK log AL a 10 HA7 The chemical shift observed for the nucleus varies between that measured at the extreme acid range and that at the extreme alkaline range 5 depending on the ratio of A HA AH The data obtained are chemical shift values in units of ppm or parts per million at various pH readings These values need to be fitted to a single ionization curve where the start and end chemical shift values and the pK are the unknown parameters 237 GraFit User s Guide 238 Fitting the Data The example data are in the file pH Titration
164. fitting result in term So for example if there are 3 fitting results and 2 different parameters the following values will be copied back to Excel Param 1 fit 1 Error 1 fit 1 Param 1 fit 2 Error 1 fit 2 Param 1 fit 3 Error 1 fit 3 Param 2 fit 1 Error 2 fit 1 Param 2 fit 2 Error 2 fit 2 Param 2 fit 3 Error 2 fit 3 The first column lists the names of the parameter values 306 Chapter 24 Passing Data Between Applications In many cases it will only be necessary to transfer a single set of results using the Results 1 part of the table If required however up to three sets of results can be copied by completing the Results 2 and Results 3 entries should these not be required then leave the entries blank Error Checking The final column of the table provides some error checking to confirm that the entries are valid Ensure that all entries read OK e Data Entry Error Checking Indicates any errors in data entries 4 COLUMN UPDATES AUTOMATICALLY El OK a P j Specifying the Results locations Ok forthe first results region on the 2 GF settings page OK 3 OK 4 f GraFit analysis page does not exist e ua al a g ee P mmm P S oat y Ensuring that the Fit is marked as Invalid when Data are Altered The final set of entries on the GF settings page is used to ensure that if the raw data within the Excel file changes perhaps because new data values have been pasted in the GraFi
165. fter the initial activation the License Administrator will receive an email with the subject License Control Panel Information telling them how to create an online account that allows them to monitor license use Our multi user licensing is implemented using software produced by a company called softWORKZ who provide the online licensing servers This email will remind you of the multi user activation code that has been used and provide a Confirmation Key that will let you add this code to your personal License Control Panel 25 GraFit User s Guide account A single account can monitor any number of difference activation codes allowing one or more GraFit licenses to be regulated by a common administrator if required Note Most email systems are set up to move unsolicited email to a Junk email folder In the event that you cannot find your License Control Panel Information email try looking in your Junk folder in case it has been moved there Registering for a New License Control Panel Account Follow the link in the email and click on the New Account button a License Control Panel Login Windows Internet Explorer wey E https www softworkz com license index a v 4 yy Favorites y License Control Panel Login SUF TWGARKZ User Name Password License Administrator Login Resend Password Why open a softWORKZ account New Account Internet Protected Mode On fg y 100 v The Licens
166. ge Scale major increment If the box is checked this value is set automatically from the graph data Uncheck the box to specify the major increment value The major increment value controls how often scale numbers and major ticks are drawn Scale minor increment If the box is checked this value is set automatically from the graph data Uncheck the box to specify the minor increment value The minor increment value controls how often major ticks are drawn Scale y axis position If the box is checked this value is set automatically from the graph data Uncheck the box to specify the axis position which should lie in between the Start and Finish values The y axis position has been set so that it appears between the Start and Finish values If required tick marks can be drawn on the y axis line from the Y Ticks page Y axis Tick settings X axis selector X Y graphs can have multiple x axes and the scale settings for each are set individually Set the x axis number from this box when first created only a single x axis will be available Axis extent By default the Entire graph size box is checked which ensures that the axis is drawn over the whole graph width Unchecking this box allows the axis to be drawn over just a portion of the graph which can be useful when multiple axes are present Enter the Start and End values as percentages of the whole graph width The Axis break symbol setting controls whether a break mark is
167. ge VS EEE background el 7 Here Range is the maximum y range and s is a slope factor The x axis represents the concentration of analyte Data fitted to this equation are usually displayed with a logarithmically scaled x axis For convenience the equation as defined attempts to provide initial estimates for the parameter values Note Estimating starting values is difficult when there are 4 unknown parameters The method used within this definition works most of the time but can give occasional poor fits Under such circumstances enter initial estimates explicitly in the non linear fit window The curve displays inhibition with increasing dose It is also possible to use the same equation for increasing response with dose In this case the y range is a negative value and the Background is the maximum response T 1C50 full 4 param show background This transformation displays the calculated background value T 1C50 full 4 param show IC50 This transformation displays the calculated background value T 1C50 full 4 param show limit This transformation displays the limiting value of the data Ligand Binding This page contains various equations describing the binding of ligands to surfaces receptors enzymes etc ac Halfman C J 1981 Methods in Enzymology 74 481 508 274 1 Site This is the simplest form of binding equation The equation is L Cap an Ka L Here y is either the amount bou
168. ge is equivalent to the Fitting Curve Style page of the XY Graph Wizard See page 163 for further information 178 Chapter 14 X Y Scatter Graphs Data Source Page fitted data only Fitted Data Properties Data source Results Fit number E Transform as Select the data fitting item to be plotted from the Results selection which lists all the available linear and non linear fits in the GraFit workspace Fitting items can hold several fits and the Fit number can be chosen The first fit is fit 1 which in most cases will be the only fit present Check the Transform as box if you wish to display the data points and fitted curve transformed as defined in the selected transformation All the transformations present in the workspace are listed See Chapter 10 for information about defining transformation items 179 GraFit User s Guide Data Source Page raw data only Data Properties Data Source Data source Data table Data X data column Substrate Y data column Rate E Transform as Select the Data table item that holds the data together with the columns that hold the x and y data Check the Transform as box if you wish to display the data points transformed as defined in the selected transformation All the transformations present in the workspace are listed See Chapter 10 for information about defining transformation items 180 Chapter 14 X Y Scatter Graphs Error Bars Page Fitted Dat
169. gether with a Quick Access Toolbar that can be customized to hold commonly used commands in a consistent manner The File menu is accessed via the GraFit logo button found in the top left of the GraFit window Using the Ribbon Bar The ribbon bar comprises a set of tabs that hold related commands Each tab has one or more ribbon groups that collect toolbar buttons Click on the button to activate the command 35 GraFit User s Guide Currently selected tab Tab specific to current item window GraFit Untitled gt E eee Table Options Ed Import Data wil E x E Select All kaal aaj Add Stock Item E OD E Edit Text Add Paste Undo Redo Cut Copy Clear Object Store Stock Item Items Clipboard Edit Object Ribbon groups The Home tab holds frequently used commands the Text tab has commands that are used for text formatting the View tab has commands that relate to the way information is displayed These three tabs are always present but in addition there are some tabs that hold commands specific to particular item windows When a data table windows is selected as above a Table tab is present on the ribbon bar when a graph item window is selected a Graph tab will be found Using the Quick Access Toolbar The Quick Access Toolbar can be used to hold any commands that you frequently access In the example below various formatting commands have been added Home Text View Click
170. gh pH e as de 5 6 7 8 9 10 T pKa min 0 High to Low Show Limit This transformation displays the limit value of the y axis at low pH T pKa min 0 High to Low Show pKa This transformation is used to represent the position of the pK on the above curve For best results set the curve resolution to High 279 GraFit User s Guide 280 2 pKa Double This equation is used when the observed parameter varies from one value at low pH to another at high pH with the variation depending upon the ionization of two groups The pK values of these groups determine the shape of the curve T pKa Double Show Curve 1 Limit Limit 10 PKaq 10 PH PKa m 4 4 Double pk H Limit 1 mite Limit 3 8 9 10 11 12 Limit Limit 10 PH PKa 1 O PH PKa 2 4 4 This transformation shows the first of the two ionization curves in isolation T pKa Double Show Curve 2 This transformation shows the second of the two ionization curves in isolation T pKa Double Show Limit 1 This transformation displays the lower limit of the double curve T pKa Double Show Limit 2 This transformation displays the middle limit of the double curve T pKa Double Show Limit 3 This transformation displays the upper limit of the double curve 2 pKa Double Bell Sometimes two ionizations determine the value of the observed param
171. guous j if iColumnStep 2 then every other column is set useful for setting xYxYxY data 323 GraFit User s Guide 324 Public sub GF SetColummNameBlockHoriz ByRef strlCalecsheet As String ByRef strGFObject As String ByRef strGFData As String ByRef data l As Variant ByVal iColumnStart As Integer ByVal iColumnStep As Integer Sets column headings within a table in an embedded GraFit object strCalcSheet strGFObject strGFData data 1ColumnStart ColumnsStep Name of sheet holding the embedded GraFit object Name of the GraFit object Name of the data table item within the GraFit object The 2D array of column names to be inserted Data will be provided by the following route Dim d As Variant d Worksheets SHEETNAME Range RANGENAME Value The data names are assumed to be in the topmost row of the specified data range The number of the data column to start setting data How many GraFit columns to increment between steps if iColumnStep 1 then the data block is contiguous if iColumnStep 2 then every other column is set useful for setting xYxXYxY data APPENDIX 1 Hard coded Equations Overview of Hard coded Equations Hard coded equation definitions use specialized pre compiled equation libraries to increase data fitting speed and or provide greater flexibility in generating initial estimates The GraFit program provides the capability to use hard coded equations though none a
172. h command You will then be given a choice of graph type to create from the Select Graph Type dialog below The resulting graph will be created using the default size If you want a graph of a specific size press the Cancel button which will result in a blank graph page Then use the New Graph tool to add a graph at a specific location and size to an existing graph item window uN Adding Graphs to an Existing Graph Item New Graph tool It is possible to create as many graphs as required on each graph item page To add an additional graph to an existing graph item window either select the New Graph command from the Graph tab which will create a graph of a default size or click the New Graph tool and drag to position the new graph where you wish it to be placed Available Graph Types Although GraFit can create a large variety of different graphs the initial choice when making a new graph is between four fundamental graph types These differ in the data that they use for example X Y Scatter Graphs hold data with separate numeric x and y values whereas a Column Chart has data values that associated with named categories The Graph gt New Graph command in legacy mode 133 GraFit User s Guide Select Graph Type XY Scatter Column Chart Bar Chart Graph a Histogram Select the basic graph type from the gallery Specific styling is then applied using the graph creation wizard which is launched when you cl
173. he following special entries can be used Entry Effect 1 All occurrences of this are replaced by the name of the untransformed x data column 2 All occurrences of this are replaced by the name of the untransformed y data column Curve Pane The Curve pane describes how fitted curves are transformed for plotting If the Transform curve box is unchecked no curve transformation is used Otherwise the remainder of the window describes the transformed curve definition Lineweaver Burk General Data Points Curve Fonts Indents 4 Transform curve X name 5 Yname F Transformed curve definition 1 y 1 Vmax S Em Vmax The Curve pane of the Transformation item Symbols double dick to insert into definition Parameters Constants Vmax Em 128 Chapter 10 Transformation Item In the sample shown above the curve transformation displays a line through the transformed data points making use of the fitted parameter values Transformed Curve Definition To draw the transformed curve which in the example above gives the linear Lineweaver Burk plot it is necessary to have an appropriate equation definition For the example that we are using the transformed equation is 1 1 Km 1 v p S Keat Eo i Kcat Eo What is being plotted is 1 v the y axis versus 1 S the x axis To show this line we therefore need an equation that is defined as E e Keat Eo Feat Eo Here x and y are the transform
174. he number of values in two selected columns and the values are updated whenever one or more data values in the source columns change A B The column holds the result of subtracting values from the second column from those in the first A B The column holds the sum of adding values in the first and second columns A B The column holds the result of dividing values in the first column by those in the second A B The column holds the product of values in the first and second columns Statistics The column types under this submenu allow the result of various statistical procedures to be displayed in the column Mean Calculate Mean Data columns 0 column s selected 71 GraFit User s Guide 72 Each row entry in the column is set to be the calculated mean of the corresponding values from the columns selected in the above dialog 2 1 X x The calculated values are updated whenever one or more values within the selected columns are modified No Data Points Each row entry in the column is set to be the number of data values N present in the corresponding rows from the selected columns This will be less than the total number of selected columns if missing data values are present Standard Deviation Each row entry in the column is set to be the calculated standard deviation of the corresponding values from the columns selected in the above dialog Standard deviation o is defined as Standard
175. here to customize The Customize button allows various aspects of the toolbar to be specified including whether the toolbar is drawn above as shown or below the ribbon bar To add commands to this toolbar either use the Customize button or right click on any command button that is found on the ribbon bar and select Add to Quick Access Toolbar Legacy style Menu and Toolbars It is also possible to run GraFit using a legacy style combination of menu and toolbars giving an appearance that is similar to GraFit version 5 and earlier The legacy style may be preferred by existing users of the program 36 Chapter 3 GraFit Basics Menu Toolbars G Grafit Untitled File Edit View Add 23 F PEETS Toolbar options click here to customize GraFit 7 using legacy style menu and toolbars Customizing Menus and Toolbars To customize menus and toolbars click the Toolbar Options button that is found on the right of each toolbar You can completely customize the commands that are found on each toolbar and menu by dragging and dropping the various command button icons Swapping Between Ribbons and Menu Toolbars The Appearance command is used to change the layout of the program The location of this command is found on the Options menu of the ribbon bar or the View menu when in menu and toolbar mode _ Toolbars Y Workspace Tabs Options Style Right To Left Animation Frame Theme Ribbon Font
176. his select the Results Listing command on the Graph tab and elect to display the results of your non linear fit item The Calculate Fit Data command in legacy mode 31 The Draw P Results Listing command in legacy mode 240 Chapter 21 Worked Data Fitting Examples Results Listing Results source Non inear Fit Fit number The information displayed in the results listing includes the parameter values for the fitted data but can also show various other pieces of information about the fit To customize the display double click on the results listing once it has been generated The following dialog box is displayed Results Listing Properties Fring Resuts Source Fit number 1 T Extra Information E Equation name E Weighting E Data source E Number of data points E Chi E Reduced Chi E Correlation coefficient linear fit only E Constants f present The Final Result For these data the final result is shown below Here the results listing has been added to the graph and in addition the pK value has been indicated visually using the pKa Single Show pKa transformation It is clear in this instance that the fitted curve describes the data well 241 GraFit User s Guide 9 6 9 4 9 2 9 8 8 8 6 8 4 8 2 8 Chemical Shift pH Parameter Value Std Error pKa 6 8001 0 0006 Limit1 9 6005 0 0003 Limit2 8 0015 0 0003 To use this transform
177. icense Administrator if you need to add more seats What happens to my installation if my computer is damaged or replaced Each activation is associated with an individual computer In the event that computer is no longer running a copy of GraFit then the License Administrator can remotely delete that seat from the license allowing you to reinstall GraFit on your new computer without affecting the number of available seats remaining on the license Please see your License Administrator if you need to remove an unwanted installation remotely What happens if have lost my activation code Activation codes and passwords are managed by your designated License Administrator Please see them for assistance lam the License Administrator and have lost my administration details Please contact Erithacus Sales via our web site and we will do our best to assist you Chapter 2 Installation and Activation My license has expired or is about to expire what can do to extend it Management of activation codes is done by your License Administrator They can extend the expiry time by purchasing a new license code and using this to top up the time remaining on your original multi user code Why is the license time limited don t want my software to expire Our multi user licensing model is based upon defined expiry periods coupled with web based management of installed seats It is not restricted to a particular version of GraFit and if a new vers
178. ich you can transfer and analyze data Note The sample Excel files were created with Microsoft Excel 2010 but saved in Excel 97 2003 compatible format to be compatible with Excel 2003 or later Excel File Description Inset Data This file is described above Kinetics Kinetics This file performs the equivalent fitting task to the Inset Data Kinetics file GraFit Fitter but is built using the GraFit Fitter template Linked Data This file is described above IC50 IC50 GraFit This file performs the equivalent fitting task to the Linked Data IC50 file Fitter but is built using the GraFit Fitter template 313 GraFit User s Guide Excel File Description IC50 96 well This file shows how it is possible to carry out more extensive rearrangement plate data including reorganizing data from a standard 96 well format 1C50 96 well plate Microsoft Excel Page Layout Formulas Data Review View Developer Add Ins A 3 Calibri 43 a z ji a 0 00 Conditional Format Cell ore Paste Ci E 20 im ta Comatona Format cole romat Clipboard Font Alignment Number styl EA 9475 KEELE 1 PRE 547 349 2 DEE ZE ECC 3 1175 d su sal oases a a IET IET METE IET 4 MECO E EE E EEE 5 571 dote 317 soso zuoo ciber csts l s6M 1 sso0l sea 36709 362 6 138 3619 1o187 21267 29s07 33710 35042 34498 33777 352682 344 7 an 12753 osa
179. ick the Create button After selecting the type of graph to create clicking the Create button will result in a specific graph creation wizard that allows the graph to be customized A description of the kind of graphs that can be created for each type is given below and more detailed information is found in the following chapters X Y Scatter Graph X Y Scatter Graphs have numeric ranges on both x and y axes Some of the types of graph that can be created are illustrated below 10 8 y 6 99 a gt 4 2 0 2 4 6 8 10 12 x Data This graph shows data points joined by a smooth Bezier curve 134 14 16 Rate 0 4 0 6 0 8 0 0 2 1 1 Substrate Substrate This graph shows data and a fitted curve inset is a linear transformation of the data Chapter 12 Creating Graphs 100 1 2 140 10 gt i A 190 0 5 3 80 00 100 o 8 08 T O a 80 2 5 0 6 ce 2 60 gt 40 0 4 40 3 0 2 i 20 a A TT om Tr 7 0 0 0 10 10 107 10 10 10 10 0 20 40 Inhibitor Fraction This graph shows a semi log plot with a This graph shows a single graph having multiple doubly split axis scale axis scales 0 6 100 4 a O E ial a go 7 o 04 8 4 e 3 S 5 S 2 a lt 02 10 a 0 1 l 0 2 4 6 0 2 4 6 8 10 Time Time This graph shows offset axes and error This graph shows multiple data sets fitted bars simulatiously to an equation with two independent variables Column Chart
180. idual plots 228 234 Residuals 235 242 Residuals plot 128 Results 97 105 Results Listing command 98 107 240 Ribbon Bar 35 Robust weighting 90 229 233 245 Rotation of objects 151 Row constant 84 Row Height command 54 Rows Selecting 59 S Savitzky Golay smoothing 85 86 Scaling power 174 Scatchard plot 290 Screen resolution 286 Security Warning Excel 297 Select All command 215 Selection Tool 141 Send to Back command 146 SetBlank function 83 84 Single user license 24 Size Column to Fit command 54 Software Activation 18 Software Updates 15 21 softWORKZ 25 Sort command 63 Spline curves 165 Split axes 189 Splitter box 53 Standard Curve 254 340 Standard deviation 72 236 Standard Error of the Mean 72 Statistics 71 Status Bar 89 103 109 126 Stock Items 42 Store Stock Item command 49 Student Licenses 22 Symbol syntax 112 Symbols font 331 Syntax Symbol names 112 System Requirements 17 T Table menu Column Format command 63 Column Properties command 68 Column Width command 54 Delete command 62 Ignore Data command 56 Insert command 62 Lock Column command 87 Preferences command 63 Row Height command 54 Size Column to Fit command 54 Sort Column s command 63 Technical Support See GraFit Technical Support Template files 47 49 Templates 42 51 285 Standard 285 Temporary variables 119 Text Alignment 173 Text
181. in practice it is rare to fit data where more than two independent variables are present Parameters Pane The Parameters pane is used to define the parameter values in the equation definition The parameter values are the unknowns in the equation and non linear regression allows data to be fitted in order to find optimal values for these parameters 113 GraFit User s Guide Enzyme Kinetics Constants Definition Estimates Fonts Indents General XA Variables Parameters Parameters The Parameters pane of the Equation item The organization of the Parameters pane is very similar to that of the X Variables pane and the definition of Symbol and Description names is performed in an analogous manner see the X Variables Pane section above The number of rows of information that are entered into the grid defines the number of parameters in the equation At least one parameter must be defined There is no formal limit to the number of parameters allowed in a GraFit equation definition but it is rare to find equations that involve more than four or five parameters Constants Pane 114 The Constants pane is used to define constants in the equation definition Constants are used to allow the entry of a value into an equation definition that is fixed in any single experiment but varies from experiment to experiment The presence of constants is optional but when present an equation definition can include any number of constants Wh
182. inear Fit item If more than one set of data is specified in the Settings pane each fit can be viewed by clicking the arrow buttons The File Print and File Print Print Preview commands show show the results in a more stylized manner as illustrated below GraFit Untitled XA Cial iprint gt 4 GO A A A Close Preview Untitled Linear Fit Linear Regression Analysis Untitled Linear Fit 06 November 2007 14 40 Robin Leatherbarrow alr Linear regression Using Print Preview from the Linear Fit item x Y data column y Number of data points Chi value v 1 2000 Correlation coefficient r 0 7559 Parameter Value a intercept 6000 b gradient 4000 The formatting of these results is controlled from the Number Format Fonts and Indents panes 106 Chapter 8 Linear Fit Item Showing the Fitted Results on a Graph A listing of the fitted parameter values can be displayed in a graph item window by selecting the Results Listing command on the Graph tab See Chapter 21 for further information Fonts and Indents Panes These panes control the way that the results are formatted The contents of these panes are common to several different item windows and are described in Chapter 7 2 The Draw gt Results Listing command in legacy mode 107 GraFit User s Guide 108 CHAPTER 9 Equation Item Overview of Equation Definitions Equation definitions are used for non l
183. inear fitting see Chapter 6 for more information about non linear fitting items The item is created from the main GraFit window using the Add Equation command The equation window itself contains a tabbed dialog box with several panes that are used to define the equation After defining the equation it must be compiled using the Compile command found in the Equation group of the Home tab before it can be used Each of the panes in this item window is discussed below Status Bar Indicators When an equation definition item is selected the status bar shows whether the equation is Compiled or Not Compiled Compilation status pane 5 The Equation Compile command in legacy mode 109 GraFit User s Guide General Pane 110 The general pane allows a description of the equation to be entered Enzyme Kinetics Constants Definition Estimates Fonts Indents General X Variables Parameters Description Michaelis Menten Equation Fits data to the Michaelis Menten equation where the rate is plotted as a function of the concentration of substrate A The General pane of the As 15 Equation item Initial estimates are provided by use of linear fitting using the Scatchard The description is optional but adding a text description of your equation definitions is strongly recommended As illustrated in the figure above the descriptive text allows rich formatting options with multiple font types styles sizes and colors
184. ion is released then your multi user license will also activate the new release In addition the pricing of multi user licenses factors in the reduced lifetime making this an affordable and managed method of software use However this licensing model is not suitable for all users and if you would prefer not to operate with time limited software then your should instead purchase one or more a single user licenses 33 GraFit User s Guide 34 CHAPTER 3 GraFit Basics Running GraFit The GraFit installation program will add a GraFit entry to the Programs list on your Windows Start menu When you run the GraFit program the main GraFit window will open Ribbon or Menu and Toolbars GraFit 7 uses by default an Office 2007 style ribbon bar to access the various commands The ribbon bar combines the functionality of both toolbars and menu bars which are used in more traditional Windows programs Office 2007 style Ribbon Bar GraFit logo button Click here to access the File menu Quick Access Toolbar Data GraFit Untitled Ll x Home Text View Table Options ey 7 ua Add Stock Item es mli Edit Text 1 Add Paste Undo Redo Cut Copy Clear Object gt m Store Stock Item oe Items Clipboard Edit Object GraFit 7 using the Office 2007 style ribbon bar The ribbon bar displays commands grouped on a series of tabs giving a less cluttered layout than the traditional display to
185. ion model They differ in that the first of these files shows an Eadie Hofstee transformation while the second uses a Lineweaver Burk transformation Rate Rate Contents of Competitive Inhibition sample Production of a Dixon Plot 0 Competitive Inhibition Substrate A PTI Epa ITT TT 040608 1 12141618 2 22 24 26 Rate Substrate Reduced Chi 0 0094 Parameter Value Std Error Vmax 8 9147 0 2682 Km 2 4453 0 1720 Ki 5 2035 0 2681 data file ToT TIER eh HO l l l l l 10 20 Column 7 Column 8 Column 9 Column 10 Column 11 Column 12 Column 13 0 3 6 1 2 Rate 1 Rate Chapter 21 Worked Data Fitting Examples Raw data values 8 O Rate 1 4 Rate 2 6 O Rate 3 HE Rate 4 g A Rate 5 44 _4 Rate 6 7 Rate 7 y Rate 8 2 7 Rate 9 J Rate 10 o Rate 11 0 T T l T T T Rate 12 0 2 4 6 8 Substrate Lineweaver Burk plot 0 8 5 J O 0 6 7 0 4 4 0 2 0 Po oe REA AA 0 0 2 0 4 0 6 0 8 1 1 Substrate Parameter Value Std Error Vmax 9 0873 0 1213 Km 2 5422 0 0973 Ki 1 9008 0 0710 Contents of Competitive Inhibition LB sample data file The sample data file Dixon plot analyses a set of inhibition data to a competitive fit then displays the da
186. is possible to load a pre built workspace from a template using the File gt New command See Chapter 4 for more information about using templates Accessing Help GraFit includes a complete on line help system to guide you through using the program In legacy mode these commands are File Store Stock Item and File Add Stock Item respectively 42 Chapter 3 GraFit Basics To view the help topics e From the Help menu select the Contents command y is Help menu To obtain specific help for a dialog box e Click on the Context Help button in the top right of the dialog box to display the help that is available for that dialog box Context Help menu Window Display Styles By default GraFit uses a similar window style to that of the Microsoft Office 2007 suite of programs However a variety of alternative window styles can be used if desired To change the window style use the Options Style command The various style colors are shown below Hog Data GraFit Untitled 010 j Home Tet View Table Options 3 43 Import Data B y X KH Selet All bal yj Add Stock Item cu 06 Gl Edit Text Add Paste Undo Redo Cut C cl Object bay Store Stock Item z dii SER i ei Blue Office Items Clipboard Edit Object Items 2 x Data x E Graph E Text Strings fie Notes y 2007 s ty e E Data BX 3 G AX x 1 2 a a iai ES iz E Enzyme Kinetics
187. it Symbols Font GraFit is supplied with a font that can be used to represent the various graph drawing symbols used by the program This can be useful when annotating graphs for example when labeling axes The font is called GraFit Symbols and can be used just like any other Windows font The various symbols are associated with the characters as shown in the following table ape epopepr epa i ei tjmiujo e CACA CA TACILACIEREALIEA LAS papeleo e fje je iif tee efe CALIENMAL IAEA TAE CILA 5 prov w xv 21 2394 5 2 0 8 2 88m a a y Note The GraFit symbols font does not contain any hinting information to assist reproduction on low resolution displays For this reason it will not display well on screen although it will print correctly 331 GraFit User s Guide 332 Index BMP file 154 CSV file 66 67 DIF file 66 67 EMF file 154 GFC file 49 GFF file 45 GFL file 325 GFR file 49 TIF file 154 TXT file 66 67 WMF file 154 _grnd_ function 84 119 _rnd_ function 84 119 A Academic Licenses 22 Activation Software 18 Activation FAQ 31 Adair Equation 277 Add command 39 Add Data command 186 242 Add Data Table command 53 Add menu 39 Index Equation command 109 247 Graph command 133 139 240 243 Hard coded Equation command 326 Linear Fit command 103 Non linear Fit command 89 238 248 Notepad command 131 Transformation command 125 Add Stock Item command
188. it window using the Add gt Transformation command The transformation window itself contains a tabbed dialog box with several panes that are used to define the transformation After defining the transformation it must be compiled using the Compile command found in the Transform group of the Home tab before it can be used Each of the panes in this item window is discussed below The Transformation gt Compile command in legacy mode 125 GraFit User s Guide Status Bar Indicators When an equation definition item is selected the status bar shows whether the equation is Compiled or Not Compiled Compilation status pane General Pane All transformations are associated with an equation definition item which must also be present in the GraFit workspace See Chapter 9 for more information about defining equations The general pane also allows a description of the transformation to be entered Lineweaver Burk General Data Points Curve Fonts Indents Associated equation Enzyme Kinetics Description Lineweaver Burk Transformation The Lineweaver Burk transformation is used to produce a double reciprocal plot of the data The transformation is associated with teh The General pane of the Michaelis Menten enzyme kinetics equation and is defined as Transformation item 1 1 Ks r 1 v S kE kalk The description is optional but adding a text description of your transformation definitions is str
189. izard 207 ICSO equations 273 288 289 Icon Changing 40 Icon command 40 Ignore Data command 56 Import Data command 49 66 Import Export Filters 49 Indents 100 102 107 Initial estimates 94 Initial Estimates 121 123 220 238 Insert command 62 Insert New Object command 110 215 Installation 18 Interactive Estimator 95 238 Internet 15 Interpolate between X limits 80 Items Loading and Saving 49 J Junk email 26 L Legend Box 174 Margin settings 175 338 Licence Control Panel 25 License Renewal 30 License Administrator 25 Licensing FAQ 31 Ligand binding 290 Ligand binding equations 274 Limiting x data range 93 105 Linear fitting 89 103 Linear regression 230 291 Basics 220 Lineweaver Burk plot 222 287 Linked object 40 Linked Objects 212 Linking 214 Linking objects 211 Lock Column command 87 Logarithmic scaling 170 Logical operators 117 M Magnification 65 141 Graph 142 Grid 65 Marquart algorithm 232 Mask curve option 161 Mean 71 236 Menu and Toolbars 35 Metafile 154 Michaelis Menten enzyme kinetics 287 Michaelis Menten Equation 270 Microsoft Excel 298 315 Mixed Inhibition 262 Move Back command 146 Move Forward command 146 Multiple axes 189 Multiple Axes 194 Multiple Regression 222 251 Advantages 224 Disadvantages 224 Plotting results 226 Uses 223 Multiple x variables 91 113 Multi u
190. l computer live update might be a little slow The example file therefore contains a check box Auto Update that controls whether updates are made continuously If Auto Update is not checked the embedded GraFit graph is only updated when the Fit Data button is clicked Getting Data Indirectly In the example above data values are typed directly into the data table and then act as the source of data used by GraFit More generally however you will want to use data that has been manipulated within Excel For example the data might come from another program or datasheet entirely or be processed within Excel to perform averaging or background subtraction etc The sample file Linked data IC50 shows how it is possible to take data from a separate Excel worksheet page and perform the equivalent analysis to that of the GraFit template file C 50 Chapter 24 Passing Data Between Applications Sort amp Find amp lt 2 Filter Select W0JOou0psOoNA Data are present within the Raw Data sheet of the file 5 Sort amp Find amp 2 Filter Select os y IO APA tea tt 1 z Y Fit Data a 5 IV Auto Update Sample datafile ed gangs The values in the Raw roma eren page can be incor d a ak Data worksheet are the data fitted and the valuesretumned transferred to the Calc Toupdate the worksheet for analysis calculations enter data into the Raw Data she
191. l is selected the mouse pointer displays as an arrow Clicking on a drawing object will select it allowing moving or resizing Clicking on the graph background and dragging will allow selection of a number of items which can also be achieved by clicking on items while holding down the SHIFT key All Other Tools Use of all other drawing tools follows a simple procedure The tool is selected which results in the mouse cursor being shown as a cross Click the left mouse button where you wish the element to be drawn and drag until it is the correct size When the button is released the element is drawn using the current fill line and font settings these can be specified see later Creating graphs using the Graph Wizard will be discussed in the next chapter Magnification The magnification of the graph can be adjusted to allow more or less of the page to be visible Magnification is controlled by the Zoom command found on the View tab Note that Zoom only affects the on screen appearance Using the Zoom Slider on the Status Bar It is also possible to adjust the magnification of the data table text using the zoom slider found on the status bar gt The View Zoom command in legacy mode 141 GraFit User s Guide The zoom slider on the status bar Select the data table then drag the slider to adjust the magnification The magnification can be set more precisely by clicking on the zoom level text Set the Graph Magnificati
192. l values are obtained that approach the minimum Y value Apart from this mathematical inconvenience non linear regression functions in the same way as linear regression We still need to know the weighting that has to be applied to the data and the calculations still aim to minimize Y The advantage is that non linear regression may be used to analyze any function that may be written y f x i e the y data value is dependent upon some arbitrary single valued function of the x data value GraFit performs non linear regression using the method of Marquart using a numerical second order method to calculate partial differentials In this way it is possible to incorporate 5 Marquart D W 1963 J Soc Ind Appl Math 11 431 41 The Marquart method is sometimes also referred to as the Marquart Levenburg method It is perhaps best explained in reference 2 Chapter 20 Theory equations of arbitrary form For an excellent explanation of regression analysis the reader is referred to the text by Bevington Standard Errors of the Calculated Parameters As well as giving the best fit values for the parameters regression analysis can provide estimates of the standard error of each parameter GraFit calculates these errors by the matrix inversion method These estimated standard errors are useful to show how accurate the calculated parameters are As a rough guide the standard error should not be greater than about 10 of the parameter valu
193. ld not necessarily give a better fitted value than if say thirty points spread over the Chapter 5 Data Tables data range were analyzed The Subset type fills the column with a subset of the data values present in one of the other columns and provides a convenient means of reducing the number of data values for analysis Create Column Subset Source data column Retain 1 point in every 2 Select the source data column and enter a value in the Retain 1 point in every field For example choosing to retain one data point in every 10 of a data set that has 200 data values would fill the column with point 1 11 21 giving 20 data points in this column The contents of the source data column are not affected by this operation Locking a Data Column It is possible to lock a data column to prevent any further modifications being made to the values that are present To lock one or more columns e Move the data entry point to the column that you wish to lock or select a range of values encompassing the columns that you wish to lock e Choose the Lock Column command on the Table tab Repeat these actions to unlock the column s 37 The Table Lock Column command in legacy mode 87 GraFit User s Guide 88 CHAPTER 6 Non linear Fit Item Overview of Non linear Fitting A non linear fit item is used to control data fitting by non linear regression This item specifies the data source and equation to use
194. left of the parameter name If more than one set of data were analyzed in the selected fit the information for each fit is given The name of the information type can optionally be displayed next to the data value by checking the Display value name option Parameter Entry The Parameter Entry column type gives a convenient way of entering parameter values for a particular equation into a column Certain graph plotting options allow these values to be used when displaying graphs The equation to use is selected after which it is possible to enter values into the column 77 GraFit User s Guide Parameter Entry Equation single exponential Cani Data 2 5X 1 2 a Parameters a 1 Initial value 100 0000 2 Rate constant 1 0000 3 A E 6 f E 9 Ve The column display differs from normal in that the name of the parameter remains displayed on the left with the value on the right When a new parameter value is entered from the keyboard the parameter name remains but the revised value is displayed in the table Constants Entry The Constants Entry column type allows the values of any constants associated with an equation to be entered in the data table This option works in the same way as the Parameter Entry option described above but shows the constants associated with the selected equation Calculated Values The column types under this submenu allow the x or y values to be calculated using the re
195. lows data to be fitted using linear regression and the results plotted 291 GraFit User s Guide Linear Regression y 3 4167 0 3833 x 8 6 gt 4 2 0 2 4 6 8 10 X Correlation coefficient 0 7936 Parameter Value Std Error a intercept 3 4167 0 6251 b gradient 0 3833 0 1111 Exponential This template fits data to a single exponential decay function using the following equation y Ao emt A graph is created showing the fit 292 Chapter 23 Standard Templates Exponential Decay 140 120 100 80 60 40 20 0 2 4 6 X Parameter Value Sid Error Initial value 130 1490 1 2017 Rate constant 0 3799 0 0066 Exponential Offset This template fits data to a single exponential decay function that goes to a background non zero value using the following equation y Ay e background A graph is created showing the fit 293 GraFit User s Guide 294 Exponential Decay with Offset 140 120 100 80 60 40 20 0 2 4 6 xX Parameter Value Std Error Initial value 128 1367 3 3007 Rate constant 0 3947 0 0243 Offset 2 3126 3 4885 Exponential Decay Various Models This template fits a single data set to a variety of exponential decay models e Single Exponential e Single Exponential with Offset e Double Exponential e Double Exponential with Offset By comparing the fits obtained using the various models it is possible to assess whether the data justify fitting
196. lution as happens with simple moving window averaging In this smoothing procedure the underlying function is approximated by a polynomial fit applied to a window of points around each data value The degree of smoothing is therefore controlled by the window width and by the order of the polynomial applied The lower the order of the polynomial or the greater the window width the smoother the result but at the expensive of losing resolution For a polynomial order of 4 best results are obtained when the window width is between 1 and 2 times the full width at half of maximum of the desired features in the data An excellent discussion of the effects of various smoothing parameters can be found in Press W H Teukolsky S A Vetterling W T amp Flannery B P 1992 Numerical Recipes in C Cambridge University Press Note Smoothing of data should always be undertaken with care Although smoothing can improve the appearance of a set of data this improvement is cosmetic only If you are fitting data it is almost always preferable to fit to the raw data values rather than the smoothed ones Subset Data that are imported from other applications or obtained from an instrumental source such as a spectrometer often have more data values than are necessary to define the experimental curve For example the data obtained from a spectrometer may contain more than a thousand points fitting so many points would be extremely time consuming and wou
197. ly to be the same for each data point Simple weighting are proportional to the value of the y data points Proportional weighting or have been individually estimated Explicit weighting The default Simple weighting is often the most appropriate setting Data Table The data to be fitted must be in a separate data table item within the GraFit workspace Select the table that holds the data from this drop down list Data Layout The data layout specifies how the data are arranged for data fitting The default layout as shown in the figure above is that the x and y data columns are to be selected individually Chapter 8 Linear Fit Item This is used to fit a single x y data set It is also possible to specify that multiple data sets are present which will result in several sets of results being generated The available options for a standard equation having a single independent variable are given below Layout Meaning bly A single data set is present and the columns holding the x and y data values are specified individually from the X data column and Y data column entries The x and y data columns do not need to be in any particular order within the data table ya yo Multiple data sets are present although all data sets have common x data values The x and y data columns are consecutive in the data table The location of the x column is set from the X data column entry the location of the final y colu
198. m from the analysis The representation of these different data types is illustrated in the following diagram The View gt Options command in legacy mode 55 GraFit User s Guide Data 2 Text Column This is column 1 This is column 2 1 2345 Text Lab 5 4a 1 000 Another label Number Missing value Ignoring Individual Data Values It is possible to mark one or more data entries to be ignored during data fitting procedures or any calculations performed within the data table This can be useful if a data value is known to be or thought to be erroneous and you do not wish to include it in an analysis Marking the data value to be ignored is preferable to deleting it as this does not remove the data point completely allowing it to be reinstated later if required To ignore a data value select it and use the Ignore Data command on the Table tab Data 3 0000 5 0000 0014 0 l oo bo ki a Ignored data point The Table gt Ignore Data command in legacy mode 56 Chapter 5 Data Tables Numeric values that are flagged to be ignored as treated as Missing Data during any data analysis procedures To restore the data value select it and repeat the Ignore Data command Moving the Data Entry Position When you have entered your data value you will need to move to the next data entry position by pressing TAB RETURN Or DOWN after entering the value The following keys control movement of the
199. m in the workspace File gt Page Setup command in legacy mode 41 GraFit User s Guide Page Setup NS Paper Size A4 210 x 297 mm Source Auto Select Orientation Margins millimeters eT 25 4 Portrait Left Top 25 4 J Landscape Header 7 462mm Header footer text Header amp F AI Footer Page amp P Restore Defaults E save as defaults A header and footer are printed at the top and bottom of the page respectively The header and footer text can contain special strings such as the file name or the time that the item was printed These special strings are specified by clicking on the gt button To omit the header and or footer from the output leave the entries for the Header text and or Footer text blank The font used to display the header and footer text can be selected by clicking on the Fonts button Changing the Printer To change the printer from the default click on the Printer button Stock Items Individual GraFit items can be stored for future reuse by selecting an item and using the Store Stock Item command They are reloaded using the Add Stock Item command Storing and loading of items is especially useful for saving and restoring equation definitions but can be used for any GraFit item See Chapter 4 for more information about the location of item files GraFit Templates Workspace files can be created by adding a number of items as described above Alternatively it
200. m the License The License Administrator can deactivate the software on any of the installed computers by clicking on the Yes link under the De activate entry Once deactivated the user of that computer will no longer be able to run GraFit unless they re enter a valid activation code and password Deactivation will typically take 24 hours to take effect Adding Extra Users Additional users up to the maximum specified by the license are added in the same way as the initial user The GraFit software must be installed on the user s computer and then when the software is run the multi user license code is entered into the activation information dialog box For subsequent users this will produce the following dialog box Activation Information MU 7 Activation password This software is licensed under a multi user agreement Please confirm that you wish to activate the software on this computer by entering the code s current activation password Activation Information If you have forgotten the password plase contact the dialog box for multi Administrator assigned to this actrvation code user activation codes second and subsequent Computer Description ROBIN ADVENT activations Password e Activation of the software will need the password that was specified during the first activation and which is available from the License Administrator who first activated the software Reusing Multi user Seats The seats in
201. mand 215 Insert Object command 214 Paste command 59 212 213 214 Paste From command 49 66 Paste Link command 212 Paste Special command 213 214 Properties command 146 149 165 194 Rename command 40 Reset Original Size 215 Select All command 215 Editing item names 40 Email support 15 Embedded object 40 Embedded Objects 211 Embedding and Linking 297 Embedding objects 211 Enhanced metafile 154 Enzyme inhibition Competitive 267 Mixed 267 Non competitive 268 269 Enzyme Inhibition 260 Enzyme kinetics 270 287 Ping Pong 266 Ternary Complex 266 Equation definition 109 115 116 Equation definition syntax 112 Equation menu Compile command 109 124 Equations 265 Compiling 124 Defining 246 335 GraFit User s Guide Error Messages 124 Functions 118 Multi line Definitions 119 Multiple Regression 224 Temporary variables 119 Erithacus Software Contacting 15 Error bars 181 Error Message 129 Error Messages 124 129 Errors 221 227 228 229 231 233 Estimates Interactive 95 Estimatrix 81 94 96 238 239 Linking to Data Table Entries 95 Evaluation Activation Code 20 Evaluation Mode 19 Excel Enable Macros 297 External Data 308 Passing Data 298 Security Warning 297 Exponential decay 272 292 Exponential decay with Offset 293 Export Graph command 154 F Ftest 73 235 Nested models 236 Field Code Command 149 Field Codes 147 File G
202. mation Embedded GraFit objects allow more sophisticated data communication using a process known as OLE automation this means that the GraFit object can be controlled programmatically from within an application that supports this Security Warnings When Using OLE Automation Microsoft Excel 2007 and later is very strict about the use of active content in order to prevent files from unknown sources being used for malicious purposes When you open a data file containing VBA program code as used by the OLE automation routines by default Excel will prevent this from running until you have confirmed that it is OK to do so A gt e hal gt 1c50 96 8 EE 1 ss Ciad i Home Insert Fage Layout Formulas Data Review 3 E 9 Calibri u A Security warning msie y a BSS gt A AN aie i 2007 after opening Clipboard E Font A a file containing 3 macros la Security Warning Some active content has been disabled Options Paja e rr AA pi y or es Clicking on the Options button results in the following dialog box 297 GraFit User s Guide 298 Microsoft Office Security Options Y Security Alert Macros ActiveX Macros amp Activex Macros and one or more ActiveX controls have been disabled This active content might contain viruses or other security hazards Do not enable this content unless you trust the source of this file Note The digital signature is valid bu
203. mentally different to a column chart 136 Number of Instances 12 14 16 18 20 22 24 26 28 30 Value A distribution of data Number of Instances Chapter 12 Creating Graphs Comparison of two distributions 137 GraFit User s Guide 138 CHAPTER 13 Graph Item Overview of Graph Items Graph items display a page of information that can hold one or more graphs and may also display results or contain OLE objects This item window is created from the main GraFit window using the Add Graph command The appearance of a blank graph item window at 50 magnification in landscape orientation is shown below Graph Blank graph item with grid lines displayed via the Grid Lines command on the Graph tab Controlling the Page View The orientation of the printed page is controlled using the File Print Page Setup command The magnification is set from the View tab The File gt Page Setup command in legacy mode SU ee View menu or using the Zoom selection on the toolbar when in legacy mode 139 GraFit User s Guide 140 Grid Lines By default the graph is displayed with a blank background but if required it is possible to add a background grid to help position the various items that form part of the graph This grid does not show during print preview and is not printed It can be turned on and off by the Grid Lines command on the Graph tab which toggles their display Th
204. mn is set from the Last Y data column entry The number of data sets present is determined automatically va pelra o Multiple data sets are present and the data sets have different x data values Pairs of x y data columns are present consecutively in the data table The location of the first x column is set from the First X data column entry the location of the final y column is set from the Last Y data column entry The number of data sets present is determined automatically X Data Column This entry specifies the column that holds the x data values See the Data Layout section above for further information Limiting x Data Range It can sometimes be useful to restrict data fitting to a subset of the values in the columns For example it can be useful to fit just the initial portion of a data set Checking the Limit x range box allows a lower and upper limit to be set on data that will be included in the analyses Y Data Column This entry specifies the column that holds the y data values See the Data Layout section above for further information Error Column If explicit weighting is chosen the error values are read from a separate column which is selected here Results Pane The Results pane holds a summary of the fitting results 105 GraFit User s Guide Es Linear Fit Number Format X column column Err column Number of data points Chi value EET re The Results pane of the L
205. mple in the simple linear regression calculations performed by pocket calculators Although often true this assumption is not always valid In many experiments the uncertainty varies with the magnitude of the y value such that the percentage error is constant To account for the different types of error distribution we introduce a weighting factor which is related to 1 0 and is used to multiply all elements of the above matrices Weighting Types The following types of error distribution are easily accounted for and can be selected for use in the GraFit program within the Linear Fit or Non linear Fit item windows Constant error The standard error de is the same for each data point and therefore a weighting factor of 1 is used Simple weighting This type of error is often given by instrumental variations for example a gauge may be read to an accuracy of 0 1 units etc 231 GraFit User s Guide Statistical error For statistical variation where f varies in proportion to y the weighting factor is 1 y This type of error distribution is uncommon but is found in systems where the main errors arise from sampling for example counting low numbers of radioactive disintegrations Proportional error For many experiments the percentage error is constant In this case o is proportional to y leading to a weighting factor of 1 y A typical example of such errors might be when for instance a measuring instrument is accurate
206. n Help gt About GraFit command Internet Our web site can be found at http www erithacus com and here you will find latest information about our software products In addition we post technical notes tips and answers to common problems Tip Be sure to check out http www erithacus com grafit to download the latest fixes and updates GraFit will check automatically for updates when the program starts You can also use the Help Check for Updates command to ensure you have the latest release 15 GraFit User s Guide 16 CHAPTER 2 Installation and Activation System Requirements Software GraFit version 7 requires Windows XP or later and is fully compatible with Windows Vista and Windows 7 Hardware Any computer that is capable of running the Windows operating system in use The GraFit Package The GraFit package comprises the following e GraFit program on CD ROM or via electronic download e The GraFit User s Guide this manual Depending upon the GraFit package that was purchased this guide may only be available in electronic form Note When GraFit is first run you will be able to register the product Registration is optional but will ensure that you are informed of the latest updates Installing GraFit Online Installation Download and run the installation program from the GraFit website www erithacus com grafit CD ROM Installation Insert the GraFit CD ROM into your CD or DVD drive
207. n addition it is possible to specify that the new axis is created offset from the main graph All new axes have associated axis and scale text which needs to be positioned as appropriate In addition the new axis ticks are set and scaled independently of any tick settings already present on the graph When you have made your initial choices press the Finish button to add the new axis Splitting an Existing Axis Choosing to split an existing axis from the Axis Manager will give the following page Axis Manager Split Settings Split position Split value 192 Chapter 15 Using Multiple Graph Axes The axis to be split should be selected from the list available The initial split position can be set using the slider control By default the axis is split in the middle e at 50 Use the slide to set this to an appropriate position The axis is split so that the scale for two new axes is set as below using the Split value to set the end of the first scale and the start of the second Original Axis Start Finish Split Axis Start Split value Finish After entering a suitable Split value click the Finish button to create the new axis Removing an Axis If you elect to remove an axis using the Axis Manager you will be presented with a list of all axes that can be deleted Axis Manager Remove x Axis Axis to remove Axis Scale Start Data Sets 1 0 2 D2 Any data or curves associated with a deleted axis will
208. n extended period then this will prevent license verification which will also result in apparent expiry of the license Reconnect to the internet and try again in order to rectify this How are Licenses Renewed Multi user licenses can be topped up at any time from the License Control Panel This involves the following process 1 A new multi user license is purchased typically from our online store see www erithacus com grafit 2 The activation code will be then be received as normal by email 3 Instead of using this license to activate GraFit as shown above it is used to add more time to an existing multi user installation using the License Control Panel From the License Control Panel choose the existing activation code that you wish to top up then click the Extend License button Chapter 2 Installation and Activation To extend this license enter a license extension code Extending the expiry date for an individual multi user a license License Extension Code Submit Cancel i S AA fot Enter the code for the new multi user license that you have just received The time available on the new license will be added to that of the existing one Note After extending your license it will typically take 24 hours before the new expiry date is shown correctly on the Help About dialog box However the License Control Panel information will be updated immediately License extension can be perform
209. n use and the value of y that is used To overcome this complication it is necessary to specify limits to the x values between which the data are to be calculated The Interpolate between X limits entries in the above dialog control the range between which the values are tested For example in the plot above the solution x 3 4 at y 7 5 would be correctly found by setting interpolation limits between x 2andx 4 Chapter 5 Data Tables y Values Use this column type to calculate theoretical y values corresponding to user entered x values using the fitting model described in a selected workspace item Calculated Y Values Fitting results Fit Single Cancel Fit 1 X data column Time Initial Rate Column 3 Data ee 1 2 3 4 A Type L y k E Column Initial Rate Name 1 0 0000 110 0000 106 6345 2 1 0000 60 0000 614794 3 2 0000 60 0000 61 1119 d 3 0000 45 0000 45 6357 5 4 0000 35 0000 34 3761 6 5 0000 20 0000 25 046 T g 9 10 dl 4 All fitted GraFit equations have only a single y value that corresponds to each x value and so there is no need to specify interpolation limits as in the previous column type When calculating y values it is possible to choose whether to use the fitted values or the explicit initial estimates that were used in the Estimatrix see Chapter 6 This allows the generation of a hypothetical data set which would result from the initial estimate values Wh
210. ncnnncnonnnnns 300 Using GraFit Fitter to Fit the Example File coooooccnncccoocnnnnncnncnnnnnnnnoos 300 Gra FEB RELCCON CE cacao 302 Transferring Data using Visual Basic Advanced ooccccccncconnonccnnnnnnnnnanoconnnnnnnonos 309 How Is Data Transter DONG ranita 312 Sample Data Pes Instale ia as 313 GraFit Automation Reference Advanced ooncccccnnncnnnnconocccnncnnnccnnnnnnancnnnonnnionnns 315 Using GraFit Automation with Microsoft ExcCel oooncccncconoccnnnnnnnonnnnnnnnoos 315 GraFit Automation ROUTINES sii aca 317 GraFit Helper ROUtIN8S oocccccoccnccnocnnnnnonnnnnnnononnncnnononnnnnnnnonnnnnnnnnnannnnnnnnnnnnnnnnss 320 Hard coded Equations Sian as 325 Overview of Hard coded EQquatiONS cccccccooccnnncnoncnnnnononcnnnnnnnnnnnnnnonononononnnnnnnnnnnanonoss 325 What are Hard coded Equations ococooccnccoccnccconnnnnnnnnnnnnncnnnnnnnnnonanononnnnnnnonnnss 325 Enabling Hard coded Equations cccccccsssccccsssccccseccceeeeecseeceseeeseesseeseesees 325 Inserting a Hard coded Equation Item ccccsssccccsesccceseeccceececsenscessenseesees 326 Generales atadca anita 327 Locating the Equation Library File oooooccnccoccnnccnnnnnnoncnnnnnnnnnnnononcnnanononos 327 A A A O da ecaneotes 327 Parameter SPa NE unrin A 328 Constant Panel E E TA 329 GraFit SYMDOIS FONT aaa a va retwantusutwcnenseiuseases 331 Grafit Symb Font dot 331 lo E
211. nd or is some factor proportional to it e g radioactivity absorbance etc or the Bound Chapter 22 Equations Provided 4 Binding Curve Show Capacity 2q 3 Show Kd 05 T T 0 2 4 6 8 10 number of moles bound per mole of receptor The capacity for binding ligand Cap is either an amount bound in terms of moles of ligand or a stoichiometric quantity in terms of moles of ligand per mole receptor depending upon the definition of y The dissociation constant of ligand from the receptor is K The concentration of free ligand L is the x data axis T 4 Site Scatchard Plot The Scatchard plot is the usual way to linearize binding data This display equation takes the Cap and K values calculated by fitting to a single site binding curve and draws the line for the Scatchard plot Use this display curve with the Scatchard data transformation T 1 Site Show Capacity This transformation shows the capacity on the plot T 1 Site Show Kd 1 site Bound Free Scatchard Plot This transformation shows the dissociation constant Kj For best results set the curve resolution to High when using this transformation 4 Site with background Often binding experiments have an inherent background y value This value can usually be determined separately by control experiments However such controls sometimes do not accurately reflect th
212. ndependent x variables that are used in the equation All equations must have at least one independent variable and can include more than one The Edit menu in legacy mode 111 GraFit User s Guide 112 Enzyme Kinetics Constants Definition Estimates Fonts Indents General X Variables Parameters X variables 1 Substrate The X Variables pane of the Equation item Naming Rules All name definitions for use in an equation definition are entered into a grid that has space for a Symbol and a Description entry All names must have a Symbol entry but the Description field is optional Description Entry If a Description entry is made GraFit will use this long name whenever it refers to the name for example when listing results If no Description entry is present GraFit will use the Symbol entry Description entries can incorporate any characters including spaces and punctuation characters that are illegal in Symbol entries Symbol Entry The symbol entry is the name used in the equation Definition page Symbol names must obey a number of rules in a similar way to variable names within a computer program e The name must begin with a letter and contain only letters and numbers In particular a symbol name cannot contain the space character The following names are legal symbols Fred k on x23 The following names however are invalid 2nd starts with a number k Ort contains a space Chapter 9
213. next page Pen Check this box to draw an outline around the data point The size and color of the outline can be specified Fill Check this box to fill the point with the solid color selected If Fill is not checked the data points will be transparent Mask curves Check this option to ensure that any curves on the graph are not drawn immediately adjacent to the data points 161 GraFit User s Guide Enter Curve Parameters Equation items only AY Graph Wizard Enter Curve Parameters Parameters Read from table 3 Direct entry Parameter When plotting theoretical curves this page allows the parameter values that describe the curve to be entered Read from table When this option is selected the parameter values are read from the data table item and column that are selected Any column in the data table can be selected but it is most convenient to use a column that has been specifically created to facilitate parameter entry see page 77 Direct Entry When this option is selected the parameter values are entered directly into the grid Enter Curve Constants Equation items only If the equation definition uses constants their values are entered into this page The operation of this page is analogous to that of the Enter Curve Parameters page described above 162 Chapter 14 X Y Scatter Graphs Select Fitting Curve Style Fitting and equation items only XY Graph Wizard Select Fitting Curv
214. ng The calculated function dialog box is shown below Chapter 5 Data Tables A Calculated Function Number of Rows Number From column x Data Result Update after changes to these columns ly Data al Column 3 0 column s selected The Number of Rows of data to be calculated can be a defined number or can be set to be the same as the number of entries in one of the other columns The function definition is entered into the Result field and uses the same syntax as is used for equation definitions see Chapter 9 By default the text entered into this field is used to calculate a result without the need for an explicit assignment statement However the name Result is predefined so that it is possible to assign values using a Result statement In addition to the standard arithmetic functions sin cos etc the following can be included within this definition Function Description Data x y Returns the data value present within the data table at column x row y ValidData x y Returns TRUE if a valid number is present in the data table at column x row y and FALSE otherwise SetBlank If this statement is encountered the current cell is set to be blank and the calculation skips to the next row Communi Returns the number of the current column This value is most useful in conjunction with the Data function 83 GraFit User s Guide 84 Function Description Row Returns the numb
215. ng a modern PC which should fit the data in a fraction of a second The calculated result is displayed below Green S Field J K Green C D amp Beynon R J 1982 Nucleic Acids Research 10 1411 1421 Chapter 21 Worked Data Fitting Examples Fraction 10 102 107 1 10 102 10 10 105 Rot Multiple Regression Multiple regression is used when the observed data values depend on more than one dependent x value As an example of this we will examine the data listed by Toney Kirsch These data consist of observations of the rate constant of a reaction k as a function of the pK of the group involved in the reaction and the molecular volume of this group Vol The data are described by the equation logk pP Ka V Vol c Here B Vand c are unknowns in the equation which is a multiple linear equation having two independent variables The data presented in the paper are in the file Multiple linear It is possible to fit these data by defining a new specific equation The required definitions are as follows Equation Name Multilinear kinetics X Variables Symbol Description pK pKa pKa value Vol Vol Molecular volume Parameter Symbol Description B Beta Beta V V V C G Constant Definition beta pKa V Vol C Toney M D amp Kirsch J F 1989 Science 243 1485 1488 251 GraFit User s Guide 252 The results obtained from fitting these data are Variable Value Standard Error B 0 3
216. nnnanonnnnnnnnoos 247 Defining a New Equation 2 oocccccccccononoccnnnnnncnonononnnnnnnnnonoccnnnnnnononanonnnnnnnnnnanooos 249 De TIMING the Equity 250 ENE tHE EQU OM so 250 MI Rda lol A e ere nceaaeae eee teers 251 Displaying Multidimensional Data oocccccoccnccnocnnonononnnnanononnncnnnnonononnnnnnnnnos 252 Reading Values from a Calibration Curve cccccsssscccccsesseccecsesseceeeseaeececsauaeeeesaaaees 254 Choosing an Equation to Fit the Data occcccocccnccocnnncnonnnnnonnnnnnanononnnnnnnnnnonos 254 Reading Values from the Fitted Curve ccccccoocccnncccnocnnnnnnnnonnnnnonanonononononnnnnnnanos 256 Fitting Several Data Sets with Some Parameters Common and Some Specific 256 Method Used to Share Variables between Data Sets cccccccocccncccnncnnncnonononos 257 Example Paired Denaturation CUFVES cccccooccnccnocnnonnnnnnnnnnnnnnnnnnnononennnnanenonos 259 Enzyme MIMI ITIOM Sa MPSS ds 260 Competitive Inhibition FIttiNg oooccncooccnnonocnnnnnnnnnnnonnnnnnononnnnnnnnnonanonnnnonos 260 PrOGUCTION OR a DIXON O o a o dd 261 Comprehensive Inhibition Modeling cccoocccnccoccnncnoconnconcnnnnanononnncnnnnnnonos 262 EQUATIONS Provided A cavsannnasnedinsquadcavedobucens E a 265 2 MOSS od 266 EMIZV ING MME IRION peiseai E E wan aoe 267 ENZYME KINETIC 45 casdnsssenebecnsoceduvecousbisanqescueeconstanasoadcuanennsuccasonedwsesansecsmoesausaanasteatnueronse 270 EXPONEN liti
217. nnnnnnnnnnonnnnnnnnanons 93 ESTIMO rss T 94 Linking Values to Data Table Entri8sS ccoooccnconccnnonononnnnnnnnnnonenonnnnnnononannnnanons 95 Interactive ESTIMO ai aia 95 PITTING CKE Paid 96 A A O 96 A A 97 Number Format Panini da 99 Fonts and Indents PINES aaa 100 Formatting Item WINdOWS anariiaacan aliada daa 101 GraFit User s Guide FORMS tting OPIO Sai aia 101 GCC RANO eee ee se no O O eee ne 101 A O A wennonen 102 Linear Fre MM us vevacssaveensausbonesauesueasedaseyasesesesesesasenavebs 103 OVERVIEW OF Linear FENE rt aaa 103 Status Bal INACIO Sia da 103 A e O eamciansmentcnetameee 104 EQUINOM id 104 Wena ia 104 A o PO A E eee 104 Data LO dde 104 X Data OLUN dis 105 Data COUM een a a a a a a a ind 105 Error COMA ada 105 RESUIES Pe 0 E EPEE AN area dad 105 Fontsand indents Panes iria aida 107 EQUATION Mea Aa 109 Overview of Equation DEFINITIONS Sui da 109 Status Bar Indicator Sanrio 109 E 9 ARO o oma a dessa cs uadeaasaumeet a ai 110 Changing FONT SI lesa 110 inserting OLE OD eS ida 110 Edita OLE OD ys dd ied S 111 Nana VES Pad o as 111 A a O ssawnsnesueaeeotaunane 112 NUMbDer or Vantablies e bado te T FE 113 E acer se aaa aucun a a 113 Blo GSS gaa RIE ad 114 Brena Ekon PAC ANRE usae sce sates a E AE AE E TEE E A E E 115 Equation DETINITION SYNTAX scan iceotiictaneeesod E eee A 116 Multi line Equation DefinitiONS oooocccncconocnnnnonononnnnnnononnnnnnnnnnnnononannnnnnnnoos 119 Useiot Temporar
218. nnnnonos 20 Descivatine Oral da 21 DEACTI Vall Ol ixcasnectncat cron cvaseacunwacnu E 21 Changing Computers 21 Checking tor Software WOU ale Ss sitiada 21 Uninstallne Ori ae 21 Different Sra tE ees n or ea a 22 Acidemie LICENSES tc ias 22 Comparison Of GraFit EIN Si idas 23 Single User versus Multiple User Licensing Of GraFit ccccoocccncnnccnnonoconcnnacnnonanonnnnoss 24 Activating GraFit for Single User or Multiple User Licensing cooomoconccoomoo 24 Multiple User Licensing Operation occccooccnccnocnnncnnonnnnnnonnnnnnnnononcnnnnonnnnnnannnononannnnanenss 24 Mittal ACtivatON sessies e a es o dd o 24 Setting up the License Control Panel ooccnccnccnnncnccnnnnnnnnnnnanononnncnnnnnnoncnnanonnns 25 Adding a Multi user License to your Administration ACCOUNT cccoccccnncncnncnnnn 26 Managing a Multi user License ccooccnnccnccnnnnnconncnnnnnnnononnnnnnnnnnonnnnnnannnonnnnnnnanenos 27 Adding Extra USerS ccccssccccsssecccensccccansccceusececeuscecseeeecsuseceseuseecsauecessaseeesaaees 28 Reusing Multi User Seats en E Ea a asa 28 Expiry Of Multi user Licenses oocccccccnocccnnncnononnnnnnnnnnnnnnnnnnnnnonnnnnnnnnnnnncnnnnnnannnos 29 Frequently asked Questions About Activation and Licensing oocccooccccncnncnannnnonnnns 31 IS atcaiccerictt avs r teese namscnastomsmate a uadeas sous eaadeaiso 31 GraFit User s Guide Multiple User License aia 32 GraFit Basies csi a aaaea ias 35 R
219. nsformation displays the limiting y value at the other extreme of pH T pKa Single Show pKa This transformation is used to represent the position of the pK on the general ionization curve For best results set the curve resolution to High Rate This set of equations fits data describing a first order rate equation 281 GraFit User s Guide 282 2 1st order 10 The equation for a process where the observed value increases with time is 1 ae A A e7Kt 3 a Shica tiit l l lt gd j Initial Rate Here A is the amount of A at time t A da is the maximum amount of A formed and A os ES k is the rate constant that describes the ae process T 1st order decay curve 107 7 Display Decay Curve This transformation converts the data into an exponential decay curve To ad linearize the curve display the y axis with S a logarithmic scale 3 0 1 l l 0 2 4 6 8 10 T 1st order show limit me This transformation displays the limiting value A T 1st order show initial The initial part of the full curve approximately the first 10 is linear with the slope being k A This transformation shows the initial linear portion 21 1st order offset te al If the observed y value has a background d or offset at time t 0 due to experimental dl Rate Curve Background constraints for example a starting E 4 E M
220. nt In GraFit equations this is implemented in the following manner labelName statements Goto labelName labelName can be any valid name using the same rules as apply to the names for symbol names The label may come before or after the Goto statement Note Indiscriminate use of Goto could result in an infinite loop Estimates Pane The Need for Starting Estimates Non linear regression works by taking initial estimates for the best fit parameter values and by an iterative process calculates successively better values until the best fit values are reached It is therefore necessary to provide the program with suitable starting estimates which should generally be of the same order of magnitude as the actual ones If an equation definition alone is provided the program cannot know what initial estimates are appropriate In this event when the equation is used the program will ask you to enter initial estimates before fitting the data To relieve users the inconvenience of estimating parameter values GraFit provides a means of generating these estimates automatically It should be noted that the method used is not foolproof and requires familiarity with manipulating equations It does however make data fitting simpler and can be over ridden if the estimates provided are inappropriate In addition equations can be defined without provision for automatic initial estimation Method Used to Obtain Initial Estimates The method u
221. ntial Decay Offset Number of parameters Number of data points 6 0000 12 Ch value 1 11 2614 43 Chi value 2 0 2757 14 F statistic 119 7674 Probability 0 0016 The results show that the probability that the two fits are equally appropriate is 0 0016 or 0 16 which is low This indicates that we can have reasonable confidence in the merit of including the extra parameter generally we require this value to be lt 5 but may prefer a value lt 1 to be sure Note Although the F test results have been shown incorporated into the same data table as the raw data it is also possible to create the analysis in a separate data table Using separate data tables can be preferable if you wish to keep the raw data separate from any analysis Data Fitting The column types under this submenu allow the results from data fitting to be displayed in a column or entry of parameter constants values associated with an equation This lets the results be viewed but more importantly allows the values to be used for subsequent analyses For example the results of one set of data fitting might provide the source data for subsequent fits 75 GraFit User s Guide Results This column type lists the results from data fitting Data Fitting Results Fit Fit Single Parameters Cancel Initial value J Rate constant Data amp xX 1 2 3 A Deny rests Parameter value Initial Rate Std error value 0 0000
222. ntrols the number of columns into which the data entries are arranged Data set 1 Data set 2 Data set 3 Data set 4 Data set 1 Data set 2 Data set 3 8 Data set 4 Custom Margins and Spacing 4 data entries with Number of columns set to 1 the default setting 4 data entries with Number of columns set to 2 Checking the Custom margins and spacing box allows the spacing of the elements within legend box to be set explicitly 175 GraFit User s Guide Fill Page This page sets the fill settings for the legend box Line Page This page specifies the line used to draw the surround of the legend box Location Page This page controls the position of the legend box within the graph page Data Curve Properties Data Points Page Fitted Data Properties Data Ports Ero ars As Association Appearance Style 9 0 lay VW o o Pen 08pt E e gt lt las a zt Es ara Mask curves This page is equivalent to the Data Point Style page of the XY Graph Wizard See page 161 for further information 176 Chapter 14 X Y Scatter Graphs Join Points Page Style Width Color This page is equivalent to the Join Style page of the XY Graph Wizard See page 164 for further information 177 GraFit User s Guide Fitted Curve Page fitted data and equations only Fitted Data Properties X limits check box for autolimit Start 0 End 10 Pesan This pa
223. ntry in the Text Strings data table Substrate Results not fitted Screen Size To ensure compatibility with the majority of systems the standard templates are formatted so that they display correctly when GraFit is run as a maximized window at a screen resolution of 1024 x 768 Using Your Own Templates In general we recommend that you build your own templates for common data fitting situations The advantages of building your own templates are summarized as follows e Specific data analyses can be created e Graphs can incorporate user specific features such as corporate logos or the name of the user e Graphs can be customized to use specific fonts and or styles Users with color printers may wish to incorporate color into their graphs those with monochrome printers will probably wish to create graphs in black and white Chapter 23 Standard Templates Users with high resolution displays can make best use of the available screen resolution when laying out the various windows See Chapter 4 for more information about working with template files Description of the Standard Templates The standard templates generally come in three related versions General Tab These analyses are appropriate for a single set of x y data Multiple Tab These analyses allow a number of data sets typically up to 8 to be analyzed together and comparison plots made The layout of the data in these templates
224. o 271 A ss E EE Gueea diate AAEE A E E ANE 273 o ainssi an a A T T A a Oaa 274 A A 278 A atts aaa wns E A cen atte eames acon T 281 Standard Templates E a A Na 285 About the Standard Temp lates ccccssscccsssscccessccccesceccensccceessecsescesseuseesauseeesaaees 285 ACCESSING TEMPLATES iia 285 Template LAVOUt iii AR 286 Using Your Own Templates ccccsscccccssscccessccccensccceescesaesceeseaecesseaeeessaesessees 286 Description of the Standard Templates ooocccncccnoccnnnnonancnnnononaconnnonanonnnnnnnncnnnonanos 287 ENZYME KINE CS eeaeee a sc eyeing sei atuawsstameaa Be uanatese S 287 NO 288 ESO BICK CO C Oi 289 Ligean BINGIN E eaen di 290 Linear RegresslOieecroern i aan nea a TNT O 291 A a a a aa 292 EXD OMEN ERAN OTS etiri 293 Exponential Decay Various Models sispan EE EEOSE 294 Ligand Binding Various MOdEIS occccooccccococnnccnonononononnonanononononnnnonononnnanonnnos 295 Passing Data between Applications ccsccsccsccsccsccsccnccsccsccsccscescescescescecceccnccscceccnccnces 297 Beyond Embedding and Linking ccoooccncccocnnncconnnnnnocnnonanonononcnnnnnnnnnnnannnnnnnnonnnnnos 297 GraFit User s Guide Security Warnings When Using OLE Automation ccsccccsseeeeceeeeeeeeeseees 297 Example Passing Data to and from Microsoft Excel cccccssecccesseeeeeseeeeees 298 Transferring Data using the GraFit Fitter Excel Template o cccccccocccnn
225. o be specified exactly relative to the current margin or to the edge of the page Arrows Properties Line Properties 152 Chapter 13 Graph Item Objects that can have lines with terminating ends allow the end type to be specified via the Arrows page Choose from a variety of arrow styles for each end of the line together with the end size and width The arrowhead color is set to be the same as the line color Font Properties A Arial W1 E O Arial Black Anal Narrow O Aral Rounded MT Bold O Arial Unicode MS El AvantGarde Effects E Underine C Strikeout Color Ms ABBDYYZ2 E Superscipt Z Subscript Baseline Dy pt Sample The Font page sets the font to use together with the style size and various effects including superscript and subscript options 153 GraFit User s Guide Paragraph Properties Text Properties p E Bullets This page sets the formatting for the selected paragraph s or text Exporting Graphs 154 While it is possible to insert graphs into other applications via the Clipboard for publishing purposes it is often important to create image files in a portable format The Export Graph command on the Graph tab allows graph images to be saved using a file format that can be used by other applications The following graph file types can be generated e Windows Metafile WMF e Enhanced Metafile EMF e Bitmap file BMP e Tagged Image Form
226. object s are moved but the movement is constrained to a purely horizontal or vertical direction depending upon the initial direction of the drag ALT left click drag Certain objects are normally constrained in their locations For example graph scale text is normally only allowed to be moved normal to the axis orientation Holding down the ALT key removes such constraints and allows arbitrary positioning Double left click For most objects this displays the object Properties dialog The exceptions are text objects for which this results in editing of the text and embedded objects for which this results in executing the default object action normally opening the item for editing The shape of the mouse cursor gives information about the current action or about which actions are available Cursor shape Meaning be Standard cursor This cursor is shown when the selection tool is chosen and the cursor is not over a selected object 144 Chapter 13 Graph Item Cursor shape Meaning A drawing tool other than the selection cursor has been chosen Left click where you wish the object to be positioned then drag until it is the desired size e The cursor is over a resizing handle which can be dragged to alter the shape of the element There are several different resizing cursors depending upon which edge of the object is to be resized be ie ls The cursor is over a selected object that can be moved in any direction f
227. ode that is entered GraFit activation codes are always 18 characters long with the first two letters indicating the type of license code If the first character is M then this is a multi user code if the first character is a number then this is a single user code that is specific for a particular software version For example 7XXXXXXXXXXXXXXXXX is a single user code for version 7 of GraFit whereas MxxxxxxxxXxXXXXXXXX is a Multi user code than will activate any release of GraFit from version 7 onwards Purchasing Single User or Multiple User Licenses Our online software shop allows you to purchase either single user licenses or multi user licenses Please visit www erithacus com grafit for more information Converting between Single User and Multiple User Modes Although it is unlikely that you will need to do this frequently it is possible to switch modes though you will need to have purchased appropriate licenses in order to allow this To change mode 1 Deactivate your current license by clicking on the Deactivate button on the Help About dialog box 2 Exit the program 3 Restart GraFit 4 Enter the new activation code and click the Activate button Multiple User Licensing Operation 24 Initial Activation When you first activate a copy of GraFit using a multi user activation code you will see the following Activation Information dialog box Chapter 2 Installation and Activation Activation Information MU _
228. odified by holding down the SHIFT or CTRL keys while dragging the handle It is possible to combine these effects by holding down both keys together Key Effect on dragging SHIFT The aspect ratio of the object is maintained during resizing CTRL The object remains maintains its current center position during resizing Resizing Text Objects Text objects have slightly different resizing behavior to other graph objects Text objects always wrap their text to fit so the height of a text object is determined by its width the text 143 GraFit User s Guide it contains and the current formatting Resizing a text object will therefore change the text wrapping Common Actions The following actions are common to many of the objects that are found on the graph Action Effect Left click The object under the cursor is selected and any selected objects are deselected If the cursor is not over a drawing object then any selection is removed SHIFT left click The object under the cursor is added to the current selection Right click An object specific context menu is displayed Left click drag The selected object s are moved Dragging can be stopped by right clicking or by pressing the ESCAPE key CTRL left click drag The selected object s are duplicated and a copy of these of these is moved to the new location This procedure can be stopped by right clicking or by pressing the ESCAPE key SHIFT left click drag The selected
229. of the data table that uses this calculated function is shown below Data E 92093 Ass 999 x 1 Data x 2 Data Calculated Results Calculated y pKa Mol Volume Rate Data fitting results Log Rate V Vol 10 6000 42 1000 40 0000 1 6021 Beta 0 393 3 9142 10 6000 60 9000 1 3000 0 1139 W 0 054 3 4567 10 5000 79 8000 0 2700 0 5666 Constant 0 6977 3 0141 10 6000 98 7000 0 0090 2 0458 10 0000 74 6000 0 0840 1 0757 9 5000 71 5000 0 0750 1 1079 9 2000 23 2000 21 0000 222 9 0000 64 4000 0 2570 0 5901 7 7000 70 5000 0 0106 1 9666 5 7000 71 6000 0 0073 2 1367 5 3000 51 1000 0 0500 1 3010 The result plotting logk V Vol versus pK and using a transformation definition to display the appropriate line through the data is shown below 253 GraFit User s Guide 254 Logk V Vol pKa Reading Values from a Calibration Curve A calibration curve records the variation of an observed value when an experimental condition is altered in a defined manner For example an instrumental reading may be measured for various known concentrations of a solution The standard curve that is produced can then be used to allow in this example the concentrations present in an unknown solution to be determined by extrapolating values from the curve These processes can be performed using GraFit by the following procedure 1 Fit the data to an equation that describes the observed trend 2 Use the Column Type Calc
230. oints and always pass each data point Spline Four orders of spline curve are available 3 4 5 and 6 the illustration is created using order 3 Higher orders give smoother curves Spline curves smooth out variation in the data and do not necessarily pass through the data points Width Style and Color These options control the appearance of the line that is drawn joining the points Modifying Graphs Once created graphs can be customized as required This involves selecting the appropriate part of the graph and invoking a specific property sheet by double clicking right clicking or choosing the Properties command on the Graph tab 27 The Edit gt Properties command in legacy mode 165 GraFit User s Guide Selectable Regions of a Graph The illustration below shows the various parts of a graph that can be selected For each a specific property sheet is available that controls the formatting options y axis scale Legend box Main graph region ie gata set 1 E 5 Legend ia text ci q 4 Data curve properties Z O A 4 8 x Data x axis text x axis scale y axis text Main Graph Region 166 The main graph region acts as the parent of the other selectable regions and is always visible the other parts of the graph can optionally be hidden Moving the main graph region will also move all the other child parts of the graph however the child regions can be moved individually to positi
231. olumns are to be selected individually This is used to fit a single x y data set It is also possible to specify that multiple data sets are present which will result in several sets of results being generated The available options for a standard equation having a single independent variable are given below Layout ELE Plas Te Chapter 6 Non linear Fit Item Meaning A single data set is present and the columns holding the x and y data values are specified individually from the X data column and Y data column entries The x and y data columns do not need to be in any particular order within the data table Multiple data sets are present although all data sets have common x data values The x and y data columns are consecutive in the data table The location of the x column is set from the X data column entry the location of the final y column is set from the Last Y data column entry The number of data sets present is determined automatically Multiple data sets are present and the data sets have different x data values Pairs of x y data columns are present consecutively in the data table The location of the first x column is set from the First X data column entry the location of the final y column is set from the Last Y data column entry The number of data sets present is determined automatically Equations with Multiple Independent Variables Equations that have multiple independent variables x axes need to acce
232. ometimes be useful to restrict data fitting to a subset of the values in the columns For example it can be useful to fit just the initial portion of a data set Checking the Limit X range box allows a lower and upper limit to be set on data that will be included in the analyses Y Data Column This entry specifies the column that holds the y data values See the Data Layout section above for further information Error Column If explicit weighting is chosen the error values are read from a separate column which is selected here Constants Pane Some equations are defined to include Constants the values of which are passed to the equation when fitting data If the selected equation includes Constants values these are entered into the grid found on this pane For equations that lack Constants this pane is empty Ea Non linear Fit Fitting Criteria Results Number Format Fonts Indents Settings Constants Estimatrix Link to table The Constants pane of Constant the Non linear Fit item Linking Values to Data Table Entries It is possible to obtain the constants values from entries within a data table Data table entries can be updated as a result of calculations and can even further linked to values in external data files To link values in this way click the Link to table checkbox and select the data table that holds the data plus the columns that are the source of the data values 93 GraFit User s Guide Estimatri
233. on choose the Compile command from the Equation group on the Home tab Compilation translates your definition into an internal form that can be executed rapidly During compilation the definition is comprehensively checked to ensure that the correct syntax has been used If an error is detected a message box will be displayed informing you of the mistake after pressing the OK button to continue the cursor will be placed at the most likely place of error An Error Message Box To obtain further information on the specific error click the Help button or press the F1 key Once the equation has been correctly compiled it can be used for data fitting and or graph plotting CHAPTER 10 Transformation Item Overview of Transformation Definitions Transformation definitions are used in order to allow data and curves to be displayed on a graph after application of a user defined transformation For example one use of a transformation definition would be to allow a double reciprocal plot to be displayed Transformations describe methods for rearranging data and equations that can be applied during graph drawing without the need to create a separate set of rearranged data 0 2 4 6 8 0 0 2 0 4 0 6 0 8 1 Substrate 1 Substrate An example of a transformed double reciprocal plot The transformation definition defines the way that the data points and the fitted curve are rearranged The transformation item is created from the main GraF
234. on by clicking on the zoom level text Manipulating Drawing Objects Selecting Objects Text Object 1 RR RAR NR A A A A Zoia STTTTrrrrrrtrriirrrrirrrrrrirreiit CLT Text Object ee rt ee ee AA Sa ee ee AS Text Object 1 eee eee eee eee eee AAA PS A AT A a Gacecoue auras AA Click to Selected select Object To select an object move the mouse pointer over it and left click Moving Objects To move an object or group of objects select the objects required and then either drag them to where you wish them to be placed or use the cursor keys to move them around Holding down the CTRL key while pressing the cursor keys produces smaller step sizes hold down SHIFT gives larger ones 142 Chapter 13 Graph Item Duplicating Objects If the CTRL key is held down while dragging an object a duplicate of the object will be moved to the new location Sizing Objects To resize an object first select it and then drag one of the resizing handles that are drawn in the corners of the object Solid resizing handles are drawn on objects that can be resized hollow handles are found on objects that do not permit resizing E EHEH o 2 This selected object has solid resizing handles that permit resizing a E t This selected object has hollow resizing handles and cannot be resized Graph Drag one of the resizing handles to alter the size of a graph object The resizing action can be m
235. on them This ensures that the whole graph can be moved around the page while maintaining the relative positioning of the constituent parts Note The x and y axis scales have a particular relationship with the main graph as it is usual to ensure that their numbers line up with the scale marks For this reason by default it is only possible to move the x axis scale in a vertical direction and the y axis scale in a horizontal direction This effect can be overridden by holding down the ALT key when moving the axis scale texts Chapter 14 X Y Scatter Graphs General Page AY Graph Properties Gener El Line _ X Tick XScale Y Ticks Y Seale Location Graph frame Drawing options Top Left Draw frame and axes over Bottom Right data points Visibility X axis text Y axis text X Axis Scale Y Axis Scale E Data Legend Box Graph frame Check the various boxes to draw a line on the various edges of the graph Drawing options Check this box to specify that the graph frame axes and tick marks should be drawn after the data points which will result in these items lying on top of the data points By default XY graphs have this option unchecked and column graphs have it checked Visibility The components of the graph are listed here and their visibility can be set by checking the appropriate box Individual items on the graph can also be hidden selecting the Hide Object command on the Graph tab Fill Page
236. ongly recommended As illustrated in the figure above the descriptive text allows rich formatting options with multiple font types styles sizes and colors allowed It is also possible as shown above to include any OLE object in the text In this particular example a Microsoft Equation 3 0 object has been embedded within the text Data Points Pane The Data Points pane describes how data points are transformed for plotting If the Transform data points box is unchecked no data point transformation is used Otherwise 126 Chapter 10 Transformation Item the remainder of the window describes how the data are to be rearranged when this transformation is applied Lineweaver Burk General Data Points Curve Fonts Indents V Transform data points Transformed x data Transformed y data Definition f xdata vydata Definition f xdata ydata 1 1 XData The Data Points pane of the Transformation item Default X name for graph Default Y name for graph ansformation ite 1 1 1 22 Symbols Parameters Constants Vmax Rm In the sample shown above the data transformation involves plotting the reciprocals of the x and y data Data Transformation Definitions Two transformation definitions need to be entered one each for the transformed x and y data Use the edit fields to enter the transformation definitions The rules for these transformation definitions are the same as described in Chapter
237. or a variety of different text elements specified by the Item selection To see the effect of changing the font settings use the File gt Print Print Preview command Indents Pane The Indents pane controls the organization of information for printing Ea Non linear Fit Settings Constants Estimatrix Fitting Criteria Results NumberFormat Fonts Indents Y Headings bnala Loram ipsum More information Dolar sit amii I Sub kearal information Consectatuer adipiscing k Yet more intoermaton Eli Glia corra ld Resuits enea A The Indents pane of the Non i a 123 456 7 890 2 BO linear Fit item Indent Sizes to 127m gt 1 905 cm x 5 715 cm Each of the different indent levels can be individually set To see the effect of changing the indent settings use the File Print Print Preview command 102 CHAPTER 8 Linear Fit Item Overview of Linear Fitting A linear fit item is used to control data fitting by linear regression This item specifies the data source and equation to use and also presents the results of the data fitting The item is created from the main GraFit window using the Add gt Linear Fit command The linear fit window itself contains a tabbed dialog box with several panes that are used to specify how the fit is calculated Each of these panes is discussed below Data fitting involves making appropriate selections in the Settings pane and possibly one or more of the other panes as
238. ot Shown in the previous figure is the definition of k as intercept gradient Eo and Km as 1 gradient Use the scroll bar to bring any further parameters into view The symbols intercept and gradient are predefined and represent the intercept and gradient of the rearranged linear plot 123 GraFit User s Guide Other predefined symbol names may also be used when defining initial estimates of your variables The symbol min will give the value of the minimum y data point and max the value of the maximum y data point The corresponding minimum and maximum x data points are minx and maxx respectively These values can be of benefit if you need to obtain rough estimates of limits where the limiting value can be approximated by the maximum recorded value It is also possible to enter numeric values directly as estimates In some situations rough numerical values provide suitable estimates without recourse to the more complex methods described above If the Omit values equal to zero box is checked then any data values x or y that are equal to zero will not be used during the data transformation This is useful with several transformations that involve taking logarithms or dividing by one of the data values as here If this box is not checked then a data point which happens to have a value of zero might cause an arithmetic error resulting in the estimates routine failing Compiling the Equation 124 To compile the equati
239. ple Data directory has a number of useful subroutines and functions that can be used when using automation Add this file to your VBA project File Import File command from the Visual Basic Editor The following routines are present in the file Public Function GF Parameter Byker strCalcSheet As String ByRef strGFObject As String ByRef strGFFit As String ByVal i As Integer _ ByVal fitNo As Integer As Variant Returns the value of a fitted parameter strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object strGFFit Name of the fitting item within the GraFit object E The parameter number to return start at 1 ELENO The index start at 1 of the fit in the object Public Function GF ParameterName ByRef strCales eet As String ByRef strGFObject As String ByRef SUrGFEiILt AS String _ ByVal i As Integer As String Returns the name of a parameter strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object strGFFit Name of the fitting item within the GraFit object e Y The parameter number to return start at 1 Public Function GF StandardError ByRef strCalcsneet As String ByRef strGFObject As String ByRef SUrGFFITt As String _ ByVal i As Integer ByVal fitNo As Integer As Variant Returns the standard error value of a fitted parameter strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name o
240. r 22 Equations Provided T Enzyme Kinetics es Lineweaver Burk Lineweaver Burk Plot This transformation draws the linear Lineweaver Burk plot for fitted enzyme kinetic data 1 Rate 0 5 5 1 Substrate T Enzyme Kinetics show Km This transformation displays the Michaelis constant K For Jou best results set the curve resolution to High when using 2 Kinetic Curve this transformation 7 k o o Show Vaa Kn T Enzyme Kinetics show Vmax 0 2 4 6 8 10 Displays the limiting rate V nax KeatlEol Substrate T Enzyme Kinetics show Vmax over Km This transformation displays the linear portion at the beginning of the graph where Substrate lt lt K Kinetics contaminating S Occasionally the reagents etc used in an enzyme assay will contain traces of contaminating substrate Therefore the rate will vary with added substrate S_ as y Vmax Saaal Seonl Km Saaal Scon In this equation S is the concentration of contaminating substrate con Exponential This page contains equations for calculating the rate constants of various exponential decay processes 271 GraFit User s Guide 272 2 Double Exponential ia aa If two decay processes occur simultaneously the observed values decay A Single Decay with time following the double S 47 exponential decay equation T e _
241. r Description Sub AbortAllCalculations Abort all current fitting calculations Function GetDataTables As Object Get the interface to the Data Tables Function GetFitResults As Object Get the interface to the Fitting Results Function GetVersionBuild As Long Get the Version Build number 0 0 0 Function GetVersionMajor As Long Get the Major Version number 0 0 0 Function GetVersionMinor As Long Get the Minor Version number 0 0 0 317 GraFit User s Guide 318 GraFit Graph Member Function GetVersionMod As Long Function IsBusy As Boolean Class GraFit DataTables GraFit DataTables Member Function GetCount As Long Function GetTablelndex index As Long As Object Function GetTableName pStr As String As Object Class GraFit DataTable GraFit DataTable Member Function SetColumn col As Long _array As Boolean Function SetColumnAssociatedData column As Long value1 As Double value2 As Double value3 As Double value4 As Double As Boolean Function SetColumnName column As Long name As String As Boolean Function SetData col As Long row As Long data As Boolean Function SetDataDouble col As Long row As Long value As Double As Boolean Class GraFit FitResults GraFit FitResults Member Function GetCount As Long Function GetResultindex index As Long As Object Function GetResultName pStr As String As Object Description Get the Version modification 0 0 0 Test whether GraFit is c
242. r ETE o a ARA E O O 134 A na ere er a ar ne nner ene ene eee nee nner er ncn ener 135 DION ias a 136 HIS TO Ma 136 O 139 OVERVIEW of Graph IRONS seran lacada airis 139 Controlling tne Page MW laa 139 Adding DraWing OBJ Sud de 140 Maem hcat OM aca aio 141 Manipulating Drawing ObjectS ccccssecccssseccceeecccseeeccceeeeeeseueceesauscessaueceseaeseesaages 142 Selectine ODE CES nn idilio 142 Moving OBJECTS a A eons 142 A A A 143 COMMONACHONS rada 144 Groupine ODJOCtS ici 145 AER INE OBJECES ia Ai 145 SCLIN TINE ZO sea cane eanseavaeel sa teera nee ooh odeeens 146 Setting the Object Cola ea 146 SELENE Text rONere Stadt ia da As 147 General ODJECE Properties in id 150 Fl PROM CTU Sort 150 PGP ROP SILOS On 151 Rotation Properties 151 LOCALIOMM PROD CITIES ral 152 AT FOWS PROD SITIOS errana ara ena dekaesaesanse coxniuntiadcaaued S 152 GraFit User s Guide FOE PROD CICS rana a 153 Paras rap PLODEICICS anto 154 EXPOTUNE Gras 154 XV Scatter Gran ida 157 TASA Grape MZ A e ea 157 Select Data Source Partir 157 SOU INI Ua SCOWING a tia 159 Selec TICK SEVIS a da 160 Select Data ROMESVIO ataco 161 Enter Curve Parameter ui 162 EnterCuUrve Constan reorit ana NE TNE OONA 162 Select FIRING CUVE SV Errea is 163 Select ION SMS aida ON 164 Nils RR o y A 165 Selectable Regions Of a Graph ooocccccccnncnnnnncnonononononoconononanonnnnnnnnnnnnnnnancnnnnnos 166 Main GraPM RELION aa ea wes cena eee 166 GeneraL A o a ein
243. r Start End Steps Limitl Limit Chemical Shift OK Parameter Estimated value Enter values into the Estimated value grid as you move the entry position the new values will be used to create a theoretical curve red on the plot As described in Chapter 20 the starting estimates for non linear regression should be reasonably close to the true values but it is not necessary to be too exact The program then iterates to find successively better values until a satisfactory fit is found When you are satisfied with your estimated values click OK to return these as the starting estimates on the Estimatrix page 239 GraFit User s Guide Fit the Data To fit the data choose the Fit Data command on the Home tab The results are then calculated and can be displayed using the File Print Preview command or viewed from the Results page Graph the Fit To plot a graph of the fitted data add a new graph to the workspace using the Add gt Graph command Elect to create an X Y Scatter Graph then use the newly created non linear fit item as the source of the data AY Graph Wizard Select Data Source Legend text Data set 4 Name from Y Source E Nondinear Fit Alfts Fts 1 to 1 Error column E Transform as You can use the Graph Add Data command to add more data sets to the plot E Show the Results on the Graph It is often a good idea to display the data fitting results on the graph To do t
244. re It must be stressed that visualization of clear cut plots of this kind depends upon having many data values to define the curves The dotted lines on the plots show the envelope within which all points are expected to be found Residuals can be calculated using options available in the data table see Chapter 5 Testing Goodness of Fit F Test There are situations where it is necessary to know whether a set of data is fitted better by one particular equation or another The F test is a statistical way of judging the goodness of fit by two different equations This test compares the y values obtained from the two fits and the degrees of freedom in each and calculates the probability that the fits are equally good A low probability value indicates that one of the two equations that giving the lower x2 fits the data significantly better To be significant the probability should be lower than 0 1 and preferably lower than 0 05 To perform the F test use the column type options available in the data table see Chapter 5 235 GraFit User s Guide Nested Models In the case where data have been fitted to two related models where one is an extension of the other by virtue of having one of more additional parameters the F statistic is calculated from the equation below OR ADI v2 x5 M Comparison of Equations with Equal Numbers of Parameters F Here the F statistic is calculated from the ratio of the y values Xo FA Xv
245. re not suitable for data fitting 2 The Trend is described by a Known Equation This is the situation where data fitting is most useful In this case based on knowledge of the experiment and the laws that govern it the data are described by a known equation i e y f X P1 P2 P3 In non mathematical terms the observed data y also known as the dependent variable varies as a function of some experimentally controlled value x the independent variable and one or more parameters p P Pz etc The exact form of the equation depends on the experiment and the conditions that are used it might be a simple linear equation y ax b or a more complex function In either case the purpose of data fitting is find values for the parameters in this equation that best describe the data For a simple linear equation as shown above this is done by linear regression In general however a more complex equation will apply Such data are fitted by non linear regression GraFit allows non linear equations of arbitrary complexity to be used for data fitting Chapter 19 Data Fitting Basics 3 A Systematic But Uncharacterized Trend is Present Occasionally data may be collected that show a distinct trend but no known equation describes this relationship For example an empirically determined calibration curve may be collected If the physical basis for the shape of the data is not well defined the appropriate equation is unknown In such si
246. re supplied as standard At present some equipment manufacturers use these to allow GraFit to fit very large quantities of data more quickly than would be possible with the standard equation definition items Hard coded equation items simply substitute for the normal equation definitions when fitting data by non linear regression see Chapter 9 It should be stressed however that for the vast majority of applications the normal user defined equation definition is quite sufficient What are Hard coded Equations Hard coded equations are pre compiled code library files and have a GFL file extension They are normally supplied for specialist applications it is not possible to generate them yourself If you believe that you have an application that might benefit from use of a hard coded equation library please contact Erithacus Software technical support for further information Advantages e Pre compiled code results in faster analysis e The partial derivative calculations that are intrinsic to non linear regression analysis can be performed explicitly rather than by numerical approximation resulting in greater speed and better precision Disadvantages e The definitions cannot be modified e tis not possible to write your own hard coded equations Enabling Hard coded Equations The menu commands to insert hard coded equations are not present by default and must be explicitly enabled The Options command on the View tab Options p
247. riation of y as some defined function of x The information on this pane describes the required equation in symbolic form At the top of the pane is an entry for Y name which allows the y axis to be given a Symbol name using the rules described earlier Equation Definition Syntax The equation definition is built up from the symbols representing parameters and constants if any with whatever arithmetic operators and mathematical functions are required For the example shown in this Chapter we have used the following equation Keat E0 S Km S In the Equation definition entry this becomes v keat Eo S Km S The syntax used is similar to that of a programming language and uses the rules described below Chapter 9 Equation Item Arithmetic Operators Symbol Operation Example Explanation Addition x y Add x and y Subtraction O Mee Subtract of f from lim di Multiplication x Multiply m by x Division a b52 Divide a by b52 Exponentiation 23 N Calculate 23 Assignment a Assign y to be twice the value of x Logical Operators Symbol Operation Example gt Is greater than k gt 7 lt Is less than x value lt conl ae Is greater than or equal to Leek 22501 lt lt Is less than or equal to Iga ley Smarte Is equal to 176 0 Note The symbol is used for both assignment and as a logical operator Which action is taken depends on the context Logical operators allow a comparison to be
248. rs can occur particularly if very large or very small numbers are used This is because computers only store decimal numbers with a limited numerical precision Although this should not affect most calculations it is 219 GraFit User s Guide advisable to scale any set of very large or very small values prior to performing calculations For example if a series of time values were entered as O to 10 seconds the rounding errors could be significant Entering the data as O to 1 femtoseconds may be more appropriate Types of Regression Analysis Regression acts to minimize the sum of the squares deviations of the experimental values from values calculated using some theoretical equation However the mechanics of the calculations are different for linear and non linear equations Linear Regression Linear regression is a technique that should be familiar to most scientists It is the mathematical equivalent of using a ruler to draw the best line through a series of data points to obtain values for the slope and intercept of this line The calculations needed to perform linear regression are relatively simple and are even found on many scientific calculators Polynomial Regression Polynomial regression is an extension of linear regression where the equation is y a bx cx dx ex Non linear Regression 220 Non linear regression allows data to be fitted in the more general case to any equation where the y data
249. rthacus Software GraFt Temple tem store User CA lUserst Robin App Data Roaming Erithacus Gra Fit Group CAProgramDiata Enthacus Software GraFt Atem Fil F Chapter 4 Working with Files Click on the button to change a directory setting By having two separate locations for templates it is possible to keep user specific files separate from general templates that are used by a group of users It can be useful for example to maintain a group directory on a network server Files in a template directory appear on the General tab of the File gt New dialog box Any subdirectories have special significance and result in additional tabs on this dialog clicking on the tab accesses the files in these subdirectories Note Empty subdirectories do not appear in the File New dialog box a GraFit Templates di Subdirectory 1 Je Subdirectory 2 Je Subdirectory 3 dl 4 F gt Template File Template File Template File 1 2 3 The files displayed on this dialog box are the sum of those present in the user and the group template directories The specific location of any individual file can be found by right clicking on the icon and choosing the Properties command In the event that a file with the same name is found in both the user and group directories the user file has precedence To create a new workspace file based on one of these templates select the one to use and click OK 51 GraFit User s Gui
250. s The default fill line and text colors for new graph objects can be specified by altering the color when no object is selected The new default colors will be used next time an object is added Alternatively the Preferences command on the Graph tab can be used to set these defaults This command also lets the default settings be stored for subsequent GraFit sessions Setting Text Properties Text properties are specified by commands found on the Text tab Na gt gt Graph GraFit Untitled Y f Arial 1 41 A ab IB Z U x x ab a e gt a a 113 Text Highlight f Color d The font size style alignment and orientation of the text can be specified from this toolbar Further text properties that are specific to particular objects may also available from the 62 R Properties dialog for the selected object If a text object is selected by clicking on it altering text properties such as font face size and color will alter the appearance of the entire object Finer control of text settings is possible for editable text by double clicking on the text item then selecting just a portion of the text Changes to text formatting will then apply only to the selected text Setting Default Text Properties The default font properties for new text objects can be specified by altering a property for example changing the font size when no object is selected The new defaults will be used nex
251. s aos otr aci anal o 3020 341 8 4546 11330 22830 29079 3w063 3331e 33648 32149 31564 298 9 10 isi i Enter values into yellow boxes Starting Dilution 11 IES conc factor IC50 1C50 Error Hill value Hill Error 12 Inhibitor 1 13 Inhibitor 2 Ifyour 14 Inhibitor 3 computer is 15 Inhibitor 4 slow uncheck 16 Inhibitor 5 Auto Update 17 Inhibitor 6 Auto Update and click Fit 18 Inhibitor 7 veel 19 Inhibitor 8 40000 Inhibitor 1 Inhibitor 2 Inhibitor 3 Inhibitor 4 Inhibitor 5 Inhibitor 6 Inhibitor 7 Inhibitor 8 20000 FARRER OY 39 0 01 0 1 1 10 100 1000 a Inhibitor 41 M 4 Sheeti Sheet2 Sheet3_ ta A 0 Ready P Note The online tutorials include a detailed description of how this file was generated IC50 96 well This file performs the equivalent fitting task to the IC50 96 well plate file plate GraFit but is built using the GraFit Fitter template Fitter IC50 96 well This is a version of the IC50 96 well plate GraFit Fitter file that gets its plate GraFit data from a linked file Fitter Linked 314 Chapter 24 Passing Data Between Applications Unprotecting the Files The sample files though not ones built from the GraFit Fitter template are saved in a way that prevents entry of data in inappropriate parts of the spreadsheet but allows correct data entry the relevant sheets are Protecte
252. s operation Derivative Calculation Source data column x Data y Data Column 3 Derivative order Savitzky Golay smoothing factors Window width 9 points must be odd number Polynomial order NLB It is assumed that the source data are already sorted If this is not the case you first need to sort them Calculation of derivatives for a set of data is performed after application of Savitzky Golay smoothing to the source data and so it is necessary to set the Savitzky Golay smoothing factors A full description of Savitzky Golay smoothing is found under the Smooth data processing type Series The series column type provides a convenient way of filling a column with a series of equally spaced values for example time series values Start value 85 GraFit User s Guide 86 Smooth This fills the selected column with the result of applying a Savitzky Golay smoothing function to a source data column The contents of the source column are not affected by this operation Savitzky Golay Smoothing Source data column x Data y Data Column 3 Smoothing factors Window width g points must be odd number Polynomial order 4 Y NLB It is assumed that the source data are already sorted If this is not the case you first need to sort them Savitzky Golay smoothing is probably the most effective means of smoothing a set of data in such a way that noise is removed without too much loss of reso
253. s Sepeaanseuaamen teat eaaasss 167 A acces aasoads nc csaecasaaiocelpuarnictevanaeciesceaeanead eeu cas metecaiueaine 167 LING IPO EE ics P A E EE E E A EE I ER E T 167 A TICKS P aE E aar a E E N 168 na o A 170 NM TICKS PAO sec E AE E E E A AE E E ETE AAE EE TE A 171 ro AICI ee aa ea a O A 172 POCA Ole Oe ea a 172 POS Scale REE IONS aa 172 PP eee ae eee 172 AXIS TOREO dadas 174 Ei A 174 Legend Pape ORI PO MU A eee re ee ce ne 175 o Men er nn nn ee E ne ere eee 176 O 176 EOC Oa AS CaF a cnc A 176 Data Curve PROD EIT IES arsenal nadia 176 Data POWMES PF Uco 176 POMP OMNES Pate a aca aceanusiecns E toda deeannacuen te A aesaees 177 Fitted Curve Page dota 178 Data OUER 24 E a E a A E area 179 Data Source Pa Eer ern TT OT O 180 Error Bals PASC seat accuccarciavscalagiarsaieniv seasons acunnunitenaiadteceaseadeenatadegexoeeatt een ad toreessades 181 AXIS ASSOCIATION PAB sieaa r T 183 A A anions 184 Parameters Pa eucariotas 185 EONSTAMES PASC ataco ao oaaaescs idad E atados 185 ESgena TEXT REGIONS a 186 Adding Extra Data to the Crap di 186 Set AXIS ASSOCIALIONS PIB id 186 Using Multiple Graph AXe6S ssesesesessssesececscsesesececscsesesececscsesesecececsesesecesscsesesececeeseseseceee 189 When to Use Multiple Axis Scaling rirscirrri s enoe a 189 Overlaying Data that have Different Scales oocoooonccnnnonononnnnnnnononononanonnnos 189 SOU PAK CS iii oc aicns 189 Creating and Managing Multiple AXe S
254. s that come from other source applications and as the source of OLE items that are inserted into other targets The situations where GraFit can act as container target or source are discussed below GraFit as an OLE container There are several places where it is possible to insert OLE items into GraFit GraFit Project Window The main GraFit project window can hold OLE objects which are inserted from the Clipboard using the Paste or Paste Special commands from the Home tab or created anew using the 74 The Edit Paste or Edit gt Paste Special commands in legacy mode 214 Chapter 18 Embedding and Linking Object Insert New Object command on the Home tab Objects inserted into the project window are always displayed as icons Activation of these objects will always launch their source application in a separate window Graph Window All graph windows can hold OLE objects which can be inserted from the Clipboard as usual or created using the Object Insert New Object command Objects inserted in a graph window can be moved and sized as required In place editing is available by double clicking on the item The size of the object can be reset using the Object Reset Original Size command from the Home tab Equation and Transformation Definition Windows These GraFit item windows hold textual comments which can incorporate embedded OLE objects See Chapters 9 and 10 for further details GraFit as an OLE source
255. s until a suitable tolerance is reached The criteria for defining this tolerance are determined by the program and can be specified when using GraFit Weighting Scheme Both linear and non linear regression analyses incorporate assumptions about the accuracy of the data points Normally they assume that all data points are equally accurate and so carry equal weighting in the analysis It is also possible to use other weighting schemes that give different weighting to one or more data values A full description of weighting is found in Chapter 20 Linear Regression of Transformed Data A familiar method of analyzing non linear data graphically is to rearrange the data to a linear form find the best line through the points and so derive the parameter values This is illustrated in the following example Enzyme kinetic data are described by a hyperbolic equation of the form Ax B x y This can be linearized by plotting 1 y versus 1 x as 1 1B 1 y x AA The ease of linear regression analysis makes it tempting to use in order to fit data that have been rearranged in this way However the results that are obtained are not statistically optimal This is because the regression calculations make assumptions about the error distribution that are not usually valid after transformation The distortion of the experimental errors by the above transformation is easily demonstrated in the following graphs 221 GraFit User s Guide
256. s you to choose the source and type of the data to plot Legend text The data set plotted is associated with an entry in the data legend The entry shows the symbol used to represent the data and a descriptive text By default the text used is taken from the y data column However if Name from Y is unchecked then you can enter your own 157 GraFit User s Guide 158 text into this field This text field can also contain two special entries that allow the data set number or the fit number to be inserted into the data legend Source Item Type Entry Linear non linear Fit Equation Data table 1 Replaced by the fit number Replaced by the data Replaced by the data number number eA Replaced by the data not applicable not applicable number Data set 1 A data legend showing the symbol and descriptive text Source Graphs are plotted using data that are present in one of the items found in the GraFit workspace The Source selection lists all items that contain suitable data for plotting and one of these must be selected GraFit graphs can plot three different types of plot on an X Y graph depending on the source selected Item Type Effect Data When raw data are plotted the data table item is selected and the x and y columns are selected for display The data points can optionally be joined with lines or curves If the data have been fitted to an equation it is usually better to select the data fitting item as this
257. sed is based on performing linear regression on rearranged data It is often possible to rearrange data in such a way that a linear plot is obtained with the parameter 121 GraFit User s Guide 122 values being related to the gradient and intercept As linear regression needs no initial estimates this can be used to estimate these parameter values providing the program knows the following 1 The rearrangement that is needed 2 How the parameter values relate to the gradient and intercept This process is illustrated using the previous example where the equation is Kcar S Eo Km 5 We can rearrange this to a linear form by plotting v S against v v _ kcat Eo v S Km Km This is the so called Eadie Hofstee equation Plotting v S against v and allows the slope and intercept to be found from which it is possible to calculate k as intercept gradient E and K as 1 gradient It must be stressed that this approach does not provide strictly correct values for k and K this is why we wish to use non linear regression in the first place Further information can be found in Chapter 19 The values provided are suitable however to use as initial estimates for the non linear regression analysis It is instructive to compare the initial estimates provided by this method with the final values produced by the non linear regression analysis As they are demonstrably different this illustrates the danger o
258. ser license 21 24 26 N Nested models 236 New Graph command 133 NMR Titration Curve 237 Non competitive Inhibition 262 Non linear fitting 89 Non linear regression 232 Non linear regression Basics 220 Notepad item 131 Number formatting 63 98 99 106 172 173 O Object embedding and linking 211 Object menu 146 Align command 145 Group command 145 Ungroup command 145 OLE automation 297 OLE objects 111 139 140 211 215 Options command 46 47 49 50 64 99 173 325 Options menu Style command 43 Outliers 229 233 Outlines command 140 pP Page Settings 41 Index Page Setup command 140 Paired Data Sets 256 Parameters 113 328 Passing Data 297 Automation 309 Sample Files 313 Paste command 59 60 66 212 214 215 Paste From command 49 66 Paste Special command 214 215 PDF files 18 Ping Pong Kinetics 266 pKa determination 279 Polynomial regression Basics 220 Preferences command 68 140 147 Print preview 40 Print Preview 97 98 240 Printer Changing 42 Printing 40 Properties command 68 146 147 150 165 194 195 Proxy Settings 19 Q Quick Access Toolbar 35 36 R Random numbers 84 Rate equations 281 Recently Used Files 47 Reduced chi squared 227 Regression analysis 90 220 227 Assumptions 228 Basics 219 Linear 230 Non linear 232 339 GraFit User s Guide Reset Original Size command 215 Res
259. ser s Guide Setting the Z Order Objects are drawn on the graph page in a defined order with the uppermost item overlaying ones beneath This z order can be changed for a selected object by using commands found in the Objects group of the Graph tab Command Meaning Bring to Front graph py Send to Back The selected object is brought to the front of all objects on the The selected object is placed underneath all objects on the graph This may result in the object becoming obscured by others on the page Move Forward Move Back Setting the Object Color The selected object is moved one place forward in the z order The selected object is moved one place back in the z order The fill pen and text colors of an object are specified separately Text color as well as the text formatting is set from the Text tab Pen and fill colors are set from the Graph tab a Th amp PA S Results Listing 1 iE e New Graph P ph 1 bd 4 bd Setting the line color Note Some objects notably graph objects have more complex color settings than can be made using the Color selectors For these it is possible to select other color options from the object Properties dialog this command found on the Graph tab or by right clicking on the object 61 The Object menu in legacy mode 62 The Edit gt Properties command in legacy mode 146 Chapter 13 Graph Item Setting Default Color
260. setting the column type to x n Data which gives the following dialog X n Data Column Independent data set x data set Column type label Label x 1 Data Load Default Label Y Use the placeholder 1 where you wish to display the x data set number It is necessary to have one x n Data column for each independent variable in the equation and these must have their X data set entries set to correspond to the X variables number in the equation definition See Chapter 6 for information about using this option when fitting data 69 GraFit User s Guide 70 y Data You may optionally select this column type if the data in the column are used as the y values for data fitting Designating a column to contain y data is not essential for data fitting but can be useful to document the data layout y Data with Associated Value It is common that an equation has two independent x variables Under these circumstances the most usual way of collecting data is to measure a series of y values at varying x for a fixed x then to collect a further set of y values at the next x value etc For example the variation of enzyme catalyzed reaction rate might be measured as a function of substrate concentration at a certain fixed inhibitor concentration The measurements are then repeated at the next inhibitor concentration and so on Here the second independent variable x in this example is the inhibitor concentration is th
261. sis However using robust weighting eliminates the outlier and the regression line is drawn through the majority of the data points Defining a New Equation 1 246 There are very few examples found in the literature where both raw data and calculated values are included However the paper by Roughton et al 1955 is notable because the raw data are presented with a set of calculated results Also the equation to be fitted is somewhat complex Because of this these data provide a good test for a data analysis package The Roughton paper examines the binding of oxygen to sheep hemoglobin as a function of the pressure of oxygen Hemoglobin has four non equivalent sites and the binding is described by the equation K p 2K K p 3K K K3p 4K K K3K p Saturation _ _ _ __ _ _ _ _J _ _ _ J _ _ _ __a_ _ _ _ CX 100 4 1 Kip K Kop K K Ksp K K K3K p i In this equation p is the pressure of oxygen and K to K are the equilibrium constants for the binding of four successive moles of oxygen per mole of hemoglobin The data are in the file Hemoglobin saturation Read this file in using the File Open command An interesting feature of these data is that some data points were collected with greater accuracy than others To account for this it is necessary to use Explicit weighting Roughton F J W Otis A B amp Lyster R L J 1955 Proc
262. splay the Eadie Hofstee plot 267 GraFit User s Guide 268 21 No inhibition This equation is the normal Michaelis Menten enzyme kinetics equation Non competitive Inhibition In this multidimensional equation rate is monitored versus substrate and inhibitor The equation for non competitive inhibition is E A na v aie 1 1 Ki Substrate Km S T Non competitive Eadie a Hofstee a a Use this to display the Eadie Hofstee plot a E ne 0 gt 0 2 4 6 8 10 Rate Substrate T Non competitive Lineweaver Burk This transformation displays the Lineweaver Burk plot 2 Non tight binding This equation describes standard or non tight binding inhibition Use in contrast to the tight binding inhibition equations see below Mo Ka Ulo 2 Non tight binding offset This equation describes standard or non tight binding inhibition Use in contrast to the tight binding inhibition equations v v 1 The equation assumes that inhibition does not proceed to 100 but that there is a background rate that is not being inhibited In real world situations this can happen for example due to residual impurities in the enzyme preparation If the background value is significant take care to question whether it is real Chapter 22 Equations Provided Zo back d 1 v vo background K 1 background Tight bin
263. ss multiple x data columns They therefore have a different set of data layout options Layout x n from y header Meaning yal This option is only available if there are two independent variables In these circumstances it is often the case that a series of y data values are collected at a fixed value of the second x variable For example the progress of a reaction might be collected with time at a number of different reagent concentrations The first x variable would be time the second would be reagent concentration Within the data table it is possible to associate a value with an entire column when this layout is selected the associated value is taken as the second x variable The associated value is set by setting the column type to y Data with Associated Value and entering a numeric x value as appropriate See Chapter 5 for further details The x and y data need to be arranged contiguously in the data table and multiple y data sets can be present The location of the X data column and the Last Y data column are selected 91 GraFit User s Guide Layout Meaning and from the program can work out how many data sets are present All the y data sets need to be in columns that are of the type y Data with Associated Value xin from y header aca ys x y2 oa This option is only available if there are two independent variables As with the previous layout the second x variable is obtained from
264. sults from a data fitting item 78 x Values Chapter 5 Data Tables Use this column type to calculate theoretical x values corresponding to user entered y values according to the fitting model described in a selected workspace item Calculated X Values Fitting results Fit Single Fit 1 Y data column y Initial Column 3 Data Interpolate between X limits Type From 1 Cin Name To 10 1 Initial 0 0000 1 0000 2 0000 3 0000 4 0000 5 0000 Rate 110 0000 680 0000 60 0000 45 0000 35 0000 26 0000 Cal 2 0 0434 1 0637 2 0636 3 0640 3 9377 4 7134 The fitting item that holds the results is chosen in the dialog box It is possible to use either the results from the data fitting or the parameter values used as the initial estimate values starting values only when carrying out the calculations Values are calculated for x values that correspond to the y values present in the selected Y data column entry A potential problem with calculating x values for a given y value is that some equations will have multiple solutions For example consider the curve shown below 79 GraFit User s Guide 80 10 Read the x value 8 6 gt Read the x value for y 4 4 2 0 0 2 4 6 8 X At y 4 the value of x read from the curve is 2 5 but at y 7 5 there are three x values 3 4 5 2 and 7 5 Clearly whether the curve is single or multiple valued in x depends upon the equation i
265. t strGFObject Name of the GraFit object strGFData Name of the data table item within the GraFit object data The 2D array of data to be inserted Data will be provided by the following route y Dim d As Variant T d Worksheets SHEETNAME Range RANGENAME Value IColumistart The number of the data column to start setting data iColumnStep How many GraFit columns to increment between steps if iColumnStep 1 then the data block is contiguous if iColumnStep 2 then every other column is set useful for setting xYxYxY data Public Sub GF SetColumnNameBlockVert ByRef strCalcsheet As String ByRef strGFObject As String ByRef strGFData As String ByRef data As Variant _ ByVal iColumnStart As Integer ByVal iColumnStep As Integer Sets column headings within a table in an embedded GraFit object strCalcsheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object strGFData Name of the data table item within the GraFit object dave The 2D array of column names to be inserted Data will be provided by the following route d Dim d As Variant d Worksheets SHEETNAME Range RANGENAME Value y The data names are assumed to be in the leftmost column l of the specified data range 1 ColumnStart The number of the data column to start setting data ColumnStep How many GraFit columns to increment between steps l if iColumnStep 1 then the data block is conti
266. t Formulas Data Review View Developer Add Ins B d calibri Ju rara ESS dia Conditional Forme att z i ainser ls 7 Fa a 32 3 39 S3 gt Be Format as Table 3 Delete ely bray ste Dal A 00 ves z ae lee Ea sE B 2 1550 325 Cell Style Be Format 2 Select gt Clipboard Font Alignment Number Styles Cells Editing K19 w Fe A B F G H l J c D E 1 Substrate Parameter Value Std Error 2 1 Vmax 8 3213 0 3471 4 3 5 4 V Auto Update 6 5 z 5 Enzyme Kinetics Data PA The sample Excel data 9 showing how data from Excelcan be incorporated into a GraFit graph and the values returned file Inset data Kinetics shows how it is possible to link together data in Excel and an embedded GraFit graph To update the calculations enter data values into the shaded table The x andy axis labels for the graph are taken from the headings onthe Calc sheet If Auto Update is checked then the fit is automatically updated whenever the raw data values change if unchecked then the calculations are only updated when the Fit Data button is pressed 0 02040608 1 1 Substrate Substrate Parameter Value Std Error Vmax 8 3213 0 3471 Km 2 0477 0 2488 44 gt mM Calc 3 This file is set up so that whenever the data in the Excel sheet changes the embedded GraFit graph also updates in real time For large data sets or when using a less powerfu
267. t can be pasted from other sources However a very convenient way of including data is to use the Data gt Get External Data From Text command ME 9 E a j Connections D AS f Existing Refresh ae 4 Connections All Connections E F P Using the Data Get External Data Pr ya got From Text command Excel 2010 shown though equivalent commands are available in Excel 2007 and 2003 The Excel text import Wizard allows the data to be inserted into a worksheet page in a very flexible manner see the Excel documentation for more information GraFit can use this mechanism to load up to three user specified files into the GraFit Fitter file To do this 1 2 3 4 Insert a new blank worksheet page for each of the datasets you wish to link to Create the file links using the Data Get External Data From Text command Format the worksheet in any way you wish For each linked data file enter the name of the worksheet on which the data are loaded into the Data Page cell on the GraFit page In the example above there are two linked data files on pages called Plate 1 Raw and Plate 2 Raw Chapter 24 Passing Data Between Applications 5 Use the Load Data n button for each of the linked data files in turn to load their contents Hint Data from laboratory instruments such as plate readers is routinely saved in a form that allows it to be read in this manner Fitting the Data Once the GF Set
268. t example it has been set to Differences To create a graph of the residuals on a new graph page select the Add Graph command and create an X Y Scatter Graph Create this graph using the data table as the source the Chemical Shift for the x data and the Differences column for the y data XY Graph Wizard Select Data Source lt gt Legend text Data set 31 Source Data All fits ts 1 ll to Ctrl click for multiple data sets Error column A Transform as U You can use the Graph Add Data command to add more data sets to p the plot Elect to join the points with a straight line as below Table Column Type in legacy mode 243 GraFit User s Guide AY Graph Wizard Select Join Style Join points Type Style Width Color Ga Gines The resulting graph is shown below 0 002 O Differences V O O O 0 O 0 002 pH As is clear from the shape of this plot see the discussions in Chapter 20 it was probably correct to use simple weighting with these data Ay versus x plots are easily made by plotting pH against the Differences To make Ay y versus y plots it is possible to create a new column to hold the result of dividing the Differences column by the original y data column Chemical Shift This can be created after data fitting by 244 Chapter 21 Worked Data Fitting Examples setting the Column Type of one of the next available columns to Column Combin
269. t fits are flagged to show that they are out of date and so need refitting Up to 4 regions within Excel can be watched for changes specify the name of the worksheet and the list of cells to be monitored The region s specified can include regions for example C4 F27 and combine regions and individual cells with commas for example B3 D3 F12 H1 H24 Input data regions Enter upto 4 regions that contain input data used forthe data fitting If data within these region s changes the fits are flagged as dirty to show that the data need to be re fitted Discontinuous regions are allowed for example A1 L8 D10 A12 E19 Specifying which regions of the Excel file should be monitored to see when the GraFit fit should be flagged as dirty Region for Fit invalidate o A A ee a NO A AAA GraFit Page The GraFit worksheet page is used to load any linked data fits and also to begin the data fitting 307 GraFit User s Guide 308 e D a i wd t y gt t Data Page Data file location e The GraFit w Load Data 1 C Users Robin Desktop Test DatalPlate 1 csv 3 worksheet page F Load Data 2 C Users Robin Desktop Test Data Plate 2 csv a contains the Fit 3 Data button Not loaded todos ff netted Sant aso allows Fit Data Not fitted loading linked a A A A A A a O IT E OT a Y al data Linked Data Files Data can be loaded into the Excel file in a number of ways It can be typed in manually and i
270. t on an additional computer that results in the installation count for this activation code being greater than permitted by the license then the latest activation will be allowed but all previous installations will automatically be deactivated 31 GraFit User s Guide 32 Grafit Activation A You have exceeded the activation limit allowed by your software license The current activation has been allowed but all previous A y Message shown when activations have been deactivated You can reactivate these j f up to the limit allowed by your license next time you run the activation of GraFit exceeds the software on those computers license limit for the code being used 4 Your user license permits activation on a maximum of 2 computers at the same time You can reactivate the previous installations up to the permitted license limit next time you run GraFit on those computers What happens if have lost my activation code It is your responsibility to keep your activation code safe In the event that you lose this code contact Erithacus Sales via our web site and we will do our best to assist you Multiple User Licenses Note You should also consult your License Administrator when dealing with multi user installations What happens if try to activate more copies than allowed by my license You will not be able to activate more computers than allowed by the license for the activation code you are using Please see your L
271. t the signature is from a publisher Enabling oe whom you have not yet chosen to trust in Excel 2007 and More information later Unless you File Path F afit Installation 0 _O Sample Excel Data Linked data IC50 xlsm enable the macros Signature it is not possible Signed by Erithacus Software Limited for Excel and Certificate expiration 27 04 2010 GraFit to Certificate issued by VeriSign Class 3 Code Signing 2004 CA Show Signature Details communicate potent recommended Enable this content Trust all documents from this publisher Open the Trust Center For the automation to function correctly this one time only click the Enable this content button then click OK If you do this then the next time you load the file you will again see the same security warning Using Content that has been Digitally Signed The dialog box shown above indicates that the file in question has been digitally signed in this instance by Erithacus Software Limited If you wish to trust all macros signed by Erithacus Software click the Trust all documents from this publisher button then click OK If you do this then you will no longer get warnings about files signed by Erithacus Software Note We recommend that you select the Trust all documents from this publisher for files signed by Erithacus Software Limited This will ensure that all our sample files will load without generating further warnings When producing Excel files it is possible to a
272. t time a text object is added Using Field Codes Text within graphs can contain field codes that are substituted with values such as entries from a data table This allows graph text to incorporate information from other item windows such information is updated dynamically Field codes are entered between curly braces and have the following syntax item name command parameters The first entry is that of the item window from which the information is to be obtained This name must be surrounded by quotes if the item name contains space characters The next entry is a command name which is specific to the item window type Finally there are one or more optional parameters which are specific to the command and separated by spaces The Graph Preferences command in legacy mode 6t The Text menu or on the Formatting Style toolbar in legacy mode 147 GraFit User s Guide Data Table Command AssValue ColumnName Data ITfTrue ITfFalse Result Item Command xColumn SOI errColumn Parameter s col index crol col row Go meow TEKU col row text Parameter s no no no Description Returns the associated value index for column col the lowest allowed index number is 2 which is the x value index number 3 gives the x3 value etc The parameter col is the number of the column The value returned is the name of the column within the data table The d
273. ta column For example the data can be entered as shown below Chapter 19 Data Fitting Basics Data J 3 4 x2 1 0000 x2 2 0000 x2 3 0000 y 1 y 2 y3 16 1 0000 0 0460 0 0760 0 1130 2 0000 0 0950 0 1460 0 2060 3 0000 0 1210 0 2110 0 2670 4 0000 0 1670 0 2640 0 3220 5 0000 0 1990 0 2940 0 3630 6 0000 0 2090 0 3330 0 3980 7 0000 0 2460 0 3610 0 4260 6 0000 0 2710 0 3740 0 4430 9 0000 0 2060 0 3960 0 4540 10 0000 0 3030 0 4270 0 4570 E k h SS Note that the y data each have an associated x value For this particular data set all the y data are associated with the same x data which corresponds to the Non linear Fit data layout type x n from y header See Chapter 6 for more details about the Non linear Fit window It is also possible to have a separate set of x data for each y data corresponding to the Non linear Fit data layout type x n from y header ya palya pa Case 2 The equation has several independent variables When an equation has several independent variables it is necessary to have the x Xz Xw Y data in separate columns This corresponds to the Non linear Fit data layout type xn from table y In order to distinguish the various data types it is essential that all the x data columns are set to x n Data with the n value representing the order of the x variable found in the equation definition See Chapter 5 for further details on column types Data Fitting
274. ta using a Dixon plot The data file shows how it is possible to generate complicated rearrangements of the underlying data which is needed to generate the Dixon plot 261 GraFit User s Guide Dixon Plot Competitive Fit 0 8 O S 1 0000 O S 2 0000 Ol S 3 0000 MW Ss 4 0000 A S 5 0000 os A S 6 0000 VY S 7 0000 o w S 8 0000 O S p e S E 0 4 A 7 Q O S 0 O C N oo o A SS a G 0 0 02 04 06 08 1 12 14 16 18 2 Inhibitor Parameter Value Std Error Vmax 9 0521 0 1317 Km 2 5263 0 1055 Ki 1 9418 0 0924 Contents of Dixon plot sample data file Comprehensive Inhibition Modeling The final sample data file is the most complex that we provide The Enzyme Inhibition file carries out a full analysis of four different inhibition models for the same data set allowing assessment of whether the data are best described by a Competitive a Non competitive a Mixed or an Uncompetitive inhibition mechanism The output for the sample data set used is reproduced below For this particular set of data it can be seen that a competitive inhibition model best fits the data as this produces the best fit both by eye and by examination of the reduced chi values The mixed type inhibition gives a similar fit quality but the additional parameter in this model does not significantly improve the fit The high error value of the additional K value is also a good indication that the extra paramet
275. table in an embedded GraFit object strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object strGFData Name of the data table item within the GraFit object Col The number of the data column start at 1 row The number of the data row start at 1 dataValue The data value to be inserted Public Sub GF BnsureInstanceClosed ByRer StrCalcsheet AS String ByRef strGFObject As String Ensures that this embedded GraFit object is fully closed Activated i objects run invisibly in the background If you have more than one or two embedded objects then it is recommended you close them down j when not needed in order to prevent memory becoming used up strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object Public Function GF TsGraritRunning ByRef strCalecSheert As string ByRef strGFObject As String As Boolean Returns True if this GraFit object is currently running strCalcSheet Name of sheet holding the embedded GraFit object strGFObject Name of the GraFit object Chapter 24 Passing Data Between Applications Public Sub GF SetDataBlock ByRef strtalc nest As String ByRef strGFObject As String ByRef strGFData As String ByRef data As Variant ByVal iColumnStart As Integer ByVal iColumnStep As Integer Sets data within a table in an embedded GraFit object strCalcSheet Name of sheet holding the embedded GraFit objec
276. table item that holds the imported data To create this item select the Import Data command Any file for which a filter is available can be imported into GraFit see the following section on Import Export Filters It is also possible to paste data from a file directly into an open data table To do this use the Paste From command found on the Table tab Import Export Filters In order to perform conversions between GraFit and other data types GraFit uses installable import export filter files Several standard filter types are supplied by default with GraFit and other filters may be available from certain instrument manufacturers or from Erithacus Software These filter files which have the GFR file extension are installed into GraFit from the Options command on the View tab Data Filters page Please contact Erithacus Software if you have special requirements for a specific filter file GraFit Template Files Template files are pre made workspace files that are used to speed up use of common procedures Template files differ from workspace files in the following ways 12 The File Store Stock Item command in legacy mode The File Add Stock Item command in legacy mode in legacy mode click on the New command button L1 which is found on the Standard toolbar 15 The File gt Import Data command in legacy mode The Edit Paste From command in legacy mode 13 16 49 GraFit User s Guide 50 e They are r
277. ter 21 Worked Data Fitting Examples If dataset 2 Then a A O STD OO ALE If dataset 3 Then o os ESEDVITA CAE assumes that dataset must be 4 adet O o Example Paired Denaturation Curves This technique of sharing parameters between two or more data sets is not limited to linear equations Any equation may be recast in this manner For example the equation below describes the denaturation of a protein as a function of the concentration of a denaturing 100 solvent m D AG exp Rr A 2 1 exp RT In this equation F is the fraction of the protein that is denatured D is the concentration of denaturant e g guanidinium hydrochloride or urea R is the gas constant T is the temperature AG is the stability of the protein and m is a slope factor Finding the difference in stability between two proteins is often more important than determining the absolute stability If the proteins are highly related it can be assumed that the value for m is the same for each but AG differs Using the technique described above it is possible to fit two data sets to a common m value but different AG values In this case the equation to be used can be defined as If dataset 1 Then a eo ine D o Ea Else O DE Y 0s ee ao ENGT Woe Sey a The x variable is the concentration of denaturant D the slope factor is m and the two values of AG are G1 and G2 The temperature is T and should be defined as a
278. the scale of the data axis to be specified By default the scale is set automatically and a linear data axis is chosen These can be altered as required it is also possible to adjust the scale settings later The text used for the data and category axes can also be entered 201 GraFit User s Guide Select Tick Styles Chart Wizard Select Tick Styles godak oaoa Set the initial tick styles for the graph The tick styles can be modified after the graph is created The Select Tick Styles page allows rapid selection of some of the more common axis tick styles More extensive tick style choices can be made once the graph has been created 202 Chapter 16 Column and Bar Charts Select Bar Style Chart Wizard Select Bar Style Type Fil style Foreground IA See ma Outline style Negative coloring Line Negative color Width O8pt The Select Bar Style page allows the chart bar representation to be chosen 203 GraFit User s Guide Select Data Point Style Chart Wizard Select Data Point Style Appearance Style 9 0 ay WW o O W Pen O8pt Hho ae 5 0 iene Mask curves It is possible to use data points to represent the values if required By default no data point style is selected 204 Chapter 16 Column and Bar Charts Select Join Style Chart Wizard Select Join Style It is possible to draw lines that join the points if required By default
279. tings page has been fully set up and the data loaded click the Fit Data button to start the data fitting y Fitting Data please wait 13 E During data fitting a progress dialog box is shown Click Abort Calculations if you wish to cancel the data fitting Abort Calculations Transferring Data using Visual Basic Advanced It is possible to gain more complete control over the data transfer process by programmatically defining how data is passed between Excel and GraFit This is done within Excel using Visual Basic for Applications VBA VBA is a programming language that allows complete control over the way that information is processed Note This is for advanced users only A full description of how VBA works is beyond the scope of this manual and you should consult reference material on VBA if you are unfamiliar with how to use this application programming environment During installation of GraFit a sample Excel file called Inset data Kinetics is placed in the GraFit Sample Data directory This file contains the same data and embedded graph as above but this file contains some VBA programming that links together the GraFit graph and the raw data In addition the fitted parameter values are brought back into Excel and presented as a table 309 GraFit User s Guide 310 Cn ld jm s Inset data Kinetics Microsoft Excel Cad Home Insert Page Layou
280. tions that do not define any constants as is the case for the figure above Legend Text Regions Legend text properties are controlled in the same way as normal text objects except that legend text cannot be displayed rotated See Chapter 13 for further information on text formatting Note The overall width of the data legend depends on the size of the individual legend text elements Each of these can be individually sized using the mouse Adding Extra Data to the Graph 186 Additional data sets fitted results or equations can be added to the graph by selecting the main graph region then choosing the Add Data command on the Graph tab This re invokes the XY Graph Wizard to allow further data to be added Most of pages are equivalent to those described earlier for creating new graphs although those pages that control initial scaling are not relevant and so are not displayed Set Axis Associations Page When graphs are first created they have a single x and y axis with which the selected data sets are associated However after creation it is possible that a graph may have additional axes and so the axis association needs to be specified when adding data This results in an additional Axis Associations page being added to the Wizard The Graph Add Data command in legacy mode Chapter 14 X Y Scatter Graphs AY Graph Wizard Set Axis Associations 1 Scale Oto 10 Fl 2 Scale 5 2 to 10 This is equivalent to th
281. to models that are more complex than a simple single exponential Chapter 23 Standard Templates Exponential Decay various models O y Single Single offset Double Double offset Ligand Binding Various Models This template fits a single data set to a variety of Ligand binding models e Single Site e Single Site with Background e Two Sites By comparing the fits obtained using the various models it is possible to assess whether the data justify fitting to models that are more complex than a simple single site model 295 GraFit User s Guide Ligand Binding Various Models 10 O Bound 8 Single Single back 6 Double D C gt O o 4 2 0 0 2 4 6 8 10 12 Free 296 CHAPTER 24 Passing Data between Applications Beyond Embedding and Linking In Chapter 18 we saw how to insert information from one application into another This means that it is possible to incorporate a GraFit analysis into another document for example into Microsoft Word or Microsoft Excel in such way that all the information that is needed to create the displayed graph is still present Double clicking on the embedded graph image will launch GraFit allowing the displayed graph to be customized or the data modified This form of data transfer works well if all the information used to create the graph is held exclusively within GraFit However is does not permit proper two way transfer of infor
282. tuations About the Standard Templates All the standard data fitting templates are defined so that as the data values are entered the results are calculated and data plotted in the background Accessing Templates To access one of the data fitting templates select the File New command Choose the template from the list shown 38 GE gy ad dd Enzyme Exponential Exponential IC 50 Ligand Binding Kinetics Offset E Linear Regression 285 GraFit User s Guide 286 Template Layout All the standard templates are formatted so that the data are entered into a data table that is on the left of the screen and the fitted data and results are updated in the graph on the right This graph is plotted as the data are being typed into the program LS Y y a G d 2 Data GraFit Untitled Home Text View Table Options Y 43 Import Data 7 e X G4 Select A E os E ha a Gj Add Stock Item 0 Ga Edit Text 4 Add Paste Undo Redo Cut Copy Clear Object y Ea Store Stock Item y Items Clipboard Edit Object Instructions 1 Enter substrate rate data into the data table 2 The data are fitted and the graph plotted automatically Enzyme Kinetics Data The x axis name is linked to the first data column name the y axis name is taken from the second data column name The title of the graph is set from the Graph Title e
283. tuations it can be useful to fit the data to a general purpose equation such as a polynomial This will allow values to be extrapolated from the standard curve although any parameter values that are obtained will not have physical meaning The use of data fitting for this purpose is illustrated in Chapter 21 Regression Analysis Regression analysis finds the best fit line or curve through a series of data This is considered to be the one that minimizes the sums of the squares deviations of the experimental data points from the theoretical curve GraFit uses regression to allow the analysis of experimental data Practical Considerations The Errors are in the y Data It is the convention to assume that the x data values represent the independent variable and the y data values represent the dependent variable i e the x data are what is varied in the experiment for example time concentration etc and the y data are the experimentally observed values y axis X axis The vertical distance the gray lines in the above diagram between the data point and the curve or line is that which is minimized by normal regression analysis i e it is assumed that all errors are present in the y data An important aspect of experimental design is to ensure that the x data are as accurate as possible so that this assumption is valid The Data Values Have a Suitable Numerical Range A consequence of computer calculation is that rounding erro
284. ual to the number of user licenses purchased Lower cost per seat than single user licensing Yes Yes A single password is used 7 2 to install all users Yearly renewal of subscription required Yes Yes Yes 3 Yes MUXXXXXXXXXXXXXXXX MAXXXXXXXXXXXXXXXX An internet connection is required periodically to verify the license validity and test for expiry and remote 23 GraFit User s Guide Single User versus Multiple User Licensing of GraFit There are two different licensing models for GraFit single user and multi user The licensing model does not affect the functionality of the program but there are some differences regarding purchase installation and software maintenance Single User Licensing Single user licensing is the traditional way of purchasing software a separate software license is purchased for each user of the program Activation of the program requires a separate activation code serial number for each installation Multiple User Licensing Multiple user licenses are advantageous in larger workgroups or organizations A single software license is purchased which allows a far larger number of installations to be performed using the same activation code In addition there is a centralized mechanism to monitor and regulate the software use Activating GraFit for Single User or Multiple User Licensing GraFit will automatically adopt the appropriate licensing model depending on the activation c
285. ulated Values x Values or Column Type gt Calculated Values y Values commands Table tab to read values from the fitted curve The sample data file Standard curve holds the analysis described below Choosing an Equation to Fit the Data In many circumstances the relationship between the x and y data values will be well defined In these cases the data will be fitted by linear or non linear regression in the usual manner However some calibration data show a clear trend of how y varies with x but are not necessarily defined by a well characterized equation 99 Table menu in legacy mode Chapter 21 Worked Data Fitting Examples For example the data below show the absorbance reading given by varying concentrations of a compound and a straight line fit to the data Absorbance O 20 40 60 80 100 120 140 160 180 200 Concentration In theory the plot of absorbance versus concentration should be linear but the data shown above describe a shallow curve While it might be of interest to find out why this effect occurs the only important consideration here is to allow readings to be taken from the calibration curve Quite clearly fitting the data to a straight line is not suitable Under these circumstances the best recourse is to fit the data to an arbitrary equation that allows a smooth curve to be drawn through the data As there is no theoretical basis for the equation any calculated parameter values will have
286. ulated values can be altered by repeating the command or by choosing the Properties command on the Table tab 34 The Table Column Type gt Column Combinations gt A B command in legacy mode 3 The Table Column Properties command in legacy mode Chapter 5 Data Tables To reset the column type to the default non calculated settings choose Column Type General Note Even though columns that hold calculated values do not allow these values to be edited it is always possible to modify the column name entry General Column Type By default all columns are set to be of General type and hold general data values that can be edited normally Data Types The column types under this submenu are used to designate that the column holds a certain type of data which may have associated additional values The entries in the column are editable but the data held in the column may have special significance for data fitting x Data You may optionally select this column type if the data in the column are used as the x values for data fitting Designating a column to contain x data is not essential for data fitting but can be useful to document the data layout x n Data When fitting an equation that has more than one independent x variable one way to hold the data is to have n separate columns for the different x values During the data fitting GraFit needs to know which column holds which x data set This is done by
287. umental uncertainties for instance a machine reading may be only accurate to some intrinsic error independent of the magnitude of the reading Providing sufficient data points have been sampled it is possible to determine the correct weighting by examination of the residual error the experimental minus the calculated value A plot of the residuals versus the observed value Ay versus y is diagnostic of the type of error involved For simple errors by definition the residual is independent of y as is seen in the plot a If the errors are of a proportional nature the residual plot will be as plot c A plot of the relative residual Ay y versus y results in the profiles b and d for simple and proportional errors respectively and confirms the error distribution Chapter 20 Theory Constant Error r b e gt le ors o Ay On 6 Ay y a O SES OO O O o Q C6 7 20 0 O Y i So 0 i Recor o 9 aso 0 SERRE Y Pra ee ee y o 6 Go OS CS O O O 69 SO On OO O 00 De d O O K O Cs 8 a Go O O Ov Go ae OO en Cee oe i 0 0 Proportional Error c d q Loan O Ay ns Ay y BO G O Ho O OO A ee za O O O ore 08 5 gt OO 0 OoOO 9 0 097 0 OF Se SOO ees Qo e O Oo O O O OO y O O 00 O a O 6 Lo os O O 00 0 0 DO o cmo es 2000 00 e cis SAA ns ome 0 0 Plot of Residual Errors Above is shown the type of residuals plots obtained when the error is constant or proportional in natu
288. urrently calculating results Description Get the number of data table items Get data table item by entry number Get data table item by item window name Description Set the data values for a whole column from an array of data Set values for the associated data fora column x 2 x 3 x 4 x 5 Set the column name Set any data value within the table Set a numeric data value within the table Description Get the number of fitting item items linear nonlinear Get fitting item by entry number Get fitting item by item window name Class GraFit FitResult GraFit FitResult Member Function ChiSq fitNo As Long As Double Function Constant index As Long As Variant Function ConstantName index As Long As String Function CorrelationCoeff fitNo As Long As Double Function DataSource As String Function EquationName As String Function GetDataFormat As Long Function GetErrColumnName As String Function GetFitStatus As Long Function GetWeighting As String Function GetXColumnName As String Function GetYCalc fitNo As Long x As Double Function GetYColumnName As String Function IsLinearFit As Long Function IsRobust As Long Function LimitXRange As Long Function LinearFitOrder As Long Function NumConstants As Long Function NumData fitNo As Long As Long Function NumFit As Long Function NumParameters As Long Function Parameter index As Long fitNo As Long As Variant
289. ut do not copy the data itself to the target Instead the target application maintains a link to the original source file Changes to this source data file result in the information copied to the target application also changing In addition if the linked object is edited from within the target this will change the original document Because linking must always involve files it is only possible to create a link after the information in the source application has been saved to disk Source Application Target Application Linking does not copy the original data but allows the target application to maintain links to the original source file Changes to the source result in the target application being updated Using Embedding and Linking 212 Windows applications that are able to employ OLE use the convention that Edit Copy from the source program will place the necessary information on the Clipboard and Edit Paste from the target application will embed an object Therefore it is the standard that embedding is used by default To link to an object the same Edit Copy command is used in the source program but depending upon the target application the object is inserted with the Edit Paste Link or Edit Paste Special command Chapter 18 Embedding and Linking The OLE object can be activated by double clicking on its representation within the target or by accessing the object menu that resides on the Edit menu and is often
290. ve through a data set by minimizing the deviation of the data from the curve Mathematically it finds values for various unknown parameters in an equation for example the slope and intercept of a straight line that produce the smallest possible sum of the squares deviations of the experimental values from the calculated ones This quantity that is minimized is denoted by the symbol y chi squared The value of y includes the variance of the data point the variance of data point is o so that data points that have greater errors contribute less to the analysis It is calculated as Ay a paz Oj Ay is the difference between the experimental and calculated y data values the subscript i is used to indicate the ith data value A further useful statistic is y2 or the reduced chi squared This is defined as below Here v is the number of degrees of freedom which is given by v N n N is the number of data points and n is the number of variables in the equation Regression analysis is the mathematical equivalent of finding the best line by eye it has the advantage however of being non subjective It is also possible to fit the best curve through the data by non linear regression This has no manual equivalent because humans are very poor at judging accurate curvature but is equally simple mathematically using computational methods 227 GraFit User s Guide Assumptions in Regression Analysis 2
291. will also show the fitted curve Equation To plot a theoretical curve select the equation item for the curve you want Linear Fit Selecting a data fitting item linear or non linear fit results in the data being plotted together with the fitted curve This is usually the way of da Noninear Fit ca plotting data fitting results Data fitting items may contain multiple fits so when a data fitting item is selected you have the option of displaying a single fit and selecting which you require or of plotting all fits X data column Y data column When the Source is a data table item it is necessary to select which columns hold the x and y data values to be plotted For all other source types these selections are not available Chapter 14 X Y Scatter Graphs Error column Checking the Error column box allows a quick way of producing an error plot when the Source is a data table item Select the column that holds the error values from the list More extensive error plotting options are available once the graph has been produced see page 181 Transform as Check the Transform as box to plot the data after transformation using the selected transformation item All available transformations which must be present in the workspace are listed Transformation of data is only available if the Source is a data table or a non linear fit item See Chapter 10 for more information about plotting transformed data Set Initial Scaling A
292. x Pane 94 Non linear regression analysis requires approximate starting estimates for the parameter values These starting values are then improved upon by iteration until the best fit curve is calculated Some equations provide a means of generating estimates automatically for other equations it is always necessary to enter explicit estimates see Chapter 9 For the former case this pane allows supplementary estimates to be entered for the latter it is essential to enter initial estimates into the grid found on this pane Ea Non linear Fit Fitting Criteria Results Number Format Fonts Indents Constants Estimatrix Link to table The Estimatrix pane of Parameter 5 the Non linear Fit Ymax item For equations that automatically provide estimates the Estimates from matrix box needs to be checked in order to allow supplementary estimates to be entered into the grid If automatic estimation is not available then this box is always checked The grid is used for entry of estimates which can be done in one of two ways 1 Just the Start value is specified In this case the value entered is taken as the initial estimate for this parameter 2 Values are entered under the Start End and Steps columns In this case a range of estimates total number Steps is generated between the Start and End values inclusive It is possible to enter just a Start value for some parameters and a range for others Whenever a range of values is involved
293. y For normal binding to a single site n should not differ significantly from 1 0 2 Cooperative with background This is the same as the equation above but includes an unknown background level in the y axis observations i e L Cap y K L background These equations are used when the observed value varies with pH They usually involve the determination of one or more pK values from the experimental data Chapter 22 Equations Provided 2 pKa min 0 8 This equation is used when the observed parameter varies with pH from O at low pH 2 to a limiting value at high pH Examples apo een nen nas SU eee include various enzymatic activities The M 3 KOAA equation is 0 T T T Limit 10 PKa IS 10 PH PKa 1 T pKa min 0 Linearized Plot This transformation displays the line for the linearized titration curve The linearized equation is Ka y 22 4 Limit H T pKa min 0 Show Limit This transformation displays the limit value of the y axis at high pH T pKa min 0 Show pKa This transformation is used to represent the position of the pK on the curve where there is a single ionization and the starting value is O For best results set the curve resolution to High 2 pKa min 0 High to Low This equation is used when the observed a a parameter varies with pH from a limiting o E value at low pH to Oat hi
294. y Va nas gssicincwdasudersesewiatuce Gaited dele ad 119 Conditional Statement S sirar er er daa 120 Serag naL AIE AEA o 120 LOODIDE Statement aa 121 GOO Late menta eds 121 ESMALTES Nes 121 The Ne d for Starting Estimates ceca did ida iaa 121 Method Used to Obtain Initial Estimates coocccncccnncnnncnnnacnnannnnccnnacnnanona 121 THe EsStimates Paenase accaunersudeunalenaaueansadeundy sateen O S E NOTAS 123 COMPING tequila 124 TranstormationiteM o cavageastesceuuessncehes Gus seeeccunestes 125 Overview of Transformation Definitions ooocccnnnccnncnnnnnnnncnnnnonanonnnnnnancnnnnonanennnnns 125 Status Bar ICI GALONS oarn 0a 126 GSTS Ae A e seo A Ln E tetas tasgeaty 126 Data POIS Na AAA aes eae 126 Data Transtormation Definitions rers as ar dal 127 AXIS Name Settini S id 128 CUE PANG a 128 Transformed Curve DefinitiON cccooooccnnccononnnnnnnnanonnnonnnnconnnonanonnnnnnnnnnnnnnnoos 129 Fonts ana Inaents Panes bat 129 Compiling the Transformati0N cccooocccnnnonononnnnnnnnonnnnnnnnonnnnonononnnnnnnnonnnnnnnnncnnnnnnanenoss 129 Notepad HemMiininaa AA A 131 US A A oie 131 TRE Notepad tem WiInG OW susana ia 131 Alternatives to the use of Notepad Items oocccnccccoccnnnnonononnncnnnncnnnnonanonnnncnancnnononoss 131 Creatine Grape nana diia N 133 How to Create a New GraphD occccccoccnccnccnnccncnnnononononnnnnnnnnnnnnnnannnnnnnnnnnnnnnnnnnannnnnnarinnnnnos 133 Available Graph TY POS id 133 XIN Scatte
295. y by available memory The data are grouped into columns and the contents of one or more columns can be used for data fitting or graph plotting Within a column each individual cell holds a data value that may be one of three different types Data Type Description Numbers Numbers are the most common data form that will be entered into GraFit They are used to plot x y scatter plots and for data fitting by regression analysis Very small or large numbers can be entered in exponential format for example 1e7 is equal to 10000000 and 1e 23 represents 107 Note The representation of numbers particularly the symbol used for the decimal point differs from country to country By default GraFit uses the country specific settings selected for your Windows installation using the Control Panel application Regional Settings However you can override these from the Options command on the View tab Number Format page Text Text entries can be used for annotation and are also used to provide the text for the category axes of charts Text values are treated as missing data during data fitting Missing Data Experimental data often contain certain values that are not available perhaps because that particular experimental sample was the one that fell on the floor GraFit allows you to insert missing values by leaving the data entry blank All the graphing and data fitting elements of the program will recognize such points and omit the
296. y given value of Rot moles second L of nucleotides of RNA B is a background value P is the proportion of the total cDNA hybridized to the ith component and Rotos is the Rot value at which 50 of the cDNA of the ith component is hybridized In practice the data obtained may be fitted assuming three binding 249 GraFit User s Guide 250 components giving a total of seven independent variables Green et a 1 have presented a sample of such data these can be found in the file DNA hybridization Defining the Equation The equation can be defined as follows X Variables Symbol Rot Rot Parameter Symbol B B Pi P1 Rot 5 1 R1 P P2 Roty 5 2 R2 Pz P3 Roty 5 3 RO Y name Sat Definition Sat Description Rot value Description Background Proportion Component 1 ROC Uso EL Proportion Component 2 ROT Ono 42 Proportion Component 3 ROC Oo a B P1 1 exp 0 693 Rot R1 P2 1 exp 0 693 Rot R2 P3 1 exp 0 693 Rot R3 Fitting the Equation The following initial estimates are appropriate to analyze the data B 0 05 P 0 3 Rot 5 0 2 P 0 3 Roto s 2 40 0 P 0 3 Roto 53 600 0 It is interesting to note that when Green et al published their results in 1982 using an Apple Il computer and employing a Patternsearch algorithm they reported that these data were fitted in approximately two hours of calculation Compare this to the amount of time taken by GraFit usi
297. y of GraFit you can copy items between 38 Chapter 3 GraFit Basics workspaces by drag and drop or by using the commands in the Clipboard group to copy and paste items via the Windows Clipboard Each item in the workspace is represented by an icon in the Items pane GraFit Items GraFit items are initially created in a new window that residues within the main GraFit window and are identified by the presence of an icon in the Items pane Closing an item window does not delete the item it simply makes the window invisible This allows you to keep open only those items that you are currently working on Managing Items Adding New Items To add a new item to the workspace e Use the Add command on the Home tab and select the type of item that you require Switching Between Items To switch between items e Click on the window to activate or click on the icon that represents this item in the project window or use the item window selection tabs Removing Items To delete an item from the workspace e Select the icon that represents the item in the project window and choose the Clear command Item Representation When an item window is closed the contents of the item still remain present in the GraFit workspace file The item window can be reopened by double clicking on the icon which is found in the Items pane The icon that represents the item is shaded so that it indicates the current state of the item as shown

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