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1. Table Of Contents GONG Al scsicsieseatdsiisssgestevsspiescuestestessacdscstcinestivsssdacsbolecssossatesssiascestovsselacsbohecsseseateussbascestoneosecs 1 How Lo Use Tints Mamani l Hov 066 Help trom MEL isis aiii daa 2 MicroCal LEC Contact At OP tiO sariin tint talado lata 3 O a dl 4 Operational SA at 5 Instrument SPAT ONS ii 6 Peoria SPAIN aS 6 PHYSICA re 6 BET NN tddi 6 Atmospheric SPC CIC AC ONS ii A aa 6 Section 1 Introduction to the 11C200 cccssccccsssscccssscccssscccsssscccsssccessscccesscccesscees 8 ki Te Bo se 9 k2 Prism A CCCSSOTIES NG 10 Ls SN 12 PT Os 13 14 BS PETAR 14 bodde BE SIN aar NG 14 Lo BET ve 14 ES Toril ExXpernmeENiS atea aeea it 16 Wat Verve 16 CACUEDTA Toll 17 Section 2 iTC200 Software and Origin cccssscsssssccccccccsssssssssssssssssssssscsesesscees 18 228 ALC ZUG SOL Ware ENN 19 22 EXpPerimental DES tir 21 25 Advanced Experimental DEN E 23 Calculating Concentrations sverd 25 ZE Tren Craven anden 27 States PN 28 STN 30 20 SUN He 31 Za MONS 33 28 Orici Reale ime Display savne 35 29 Orie Data Analysis T totial sereine en a A E o 37 Section 3 Instrument Maintenancce cccccccssscccssscccsssccccssscccssscccssscccsscesscesscesccescs 38 Seb Gems the Cilka Syringe e n EG 39 ETER 39 o te OVER EEE EE EE EN 41 3 2 Chang me en ST RST 43 TN 45 4 Cass Pipete Tre 46 35 X axis Calibration CASO alada ainia 47 3 0 Temperature Calibra
2. k Fill both cells with distilled water at ambient temperature fill the titration syringe and insert the syringe into the sample cell Click on the Load Run File button in the Advanced Experimental Design tab choose WATER INJ and click Open Name the run and click Start The instrument will seek the run temperature equilibrate the system and begin making injections Each peak should be small with all the peaks approximately the same shape Once the run has finished and the instrument is thermostatting again open the ITC Calibrations project to analyze the data It is located in the Origin directory at C Origin70 TCCalibrations OPJ Click on the Water Water Report button A window will pop up asking which files to analyze select the file you just generated click Add Files and click OK Origin will analyze the data for several seconds Once it has finished it will ask you to save the results Give the file a unique name There should be a graph with the water DP data and a textbox Zoom in using the magnifying glass over a plus sign icon on the top toolbar so that you can read the textbox To zoom in and read the text select the magnifying glass over a plus sign icon on the top toolbar and click on the textbox Water Water Report VPITC11 06 834 12 5 2006 08 42 23 Raw DP Data 10uL injections Water Water Specifications Mean Energy Injection less than 1 5 uCal SD less than 0 15 uCal Baseline Offsets Have Been Manip
3. The Control Company meter is model 9612 the probe is model 4021 Make sure the cells are clean and load them with degassed distilled water Residue in the cells or empty cells will yield unpredictable results Insert the probe into the sample cell and turn it on 1 Use the Temperature control box under the Instrument Controls tab in the 1TC gt 0 software to set the temperature to 30 C 2 Set the Plot Idle Data flag so that the current data is displayed 3 When the instrument is thermostatting at the target temperature the DP has settled at a negative stable value and the DT is 0 use the data reader tool and the display from the meter to take temperature data points as close together as possible 4 Repeat for the second check point at 70 C Ifthe error for each is not less than 0 2 C please contact MicroCal for assistance It is recommended that users keep a log with the date of the calibration check and the software and meter readings for each temperature point
4. C Time Left fF Load Run File Save Run File Temp C DP pl al s 30 158 4 1 DP 50 0 50 100 150 200 250 300 350 400 450 500 550 600 Time sec 1 DP Tools oom 0 05 Zoom 0 5 Show All Plot Idle Data The Real Time Plot tab displays the current data If no run has been started the graph will be blank The Temp C box displays the current cell temperature The DP uCal s box displays the current DP Technically it is the reading from the sensor between the sample and reference cells This data channel shows the changes in heat in the sample cell as events occur and is plotted in this graph The three buttons at the lower left control the graph resolution all three resize the y axis relative to the last data point plotted Zoom 0 05 alters the vertical range to the last data point plus or minus 0 05uCal s Zoom 0 5 alters the range to the last data point plus or minus 0 5uCal s Show All rescales the y axis so that all the data points are visible The Plot Idle Data box when checked will display the current DP readings on the graph even though no run is being performed It is frequently helpful for troubleshooting 2 6 Setup e ITC200 Default User System ITC Options Help gt Load Run File Save Run File User Setup Data File Path C ITC2004data Setup File Path C 1TC200 setup Init Setup File Lastra gt Cells Boot Temp 30 C Ga Current User Default Us
5. displayed as will a percent error for both power and energy The reported error in deflection or energy should be less than 1 If the error 1s reported as higher than 1 please contact MicroCal For a more rigorous analysis once the calibration is done and the system is thermostatting again open the ITC Calibrations project Click on the Y Axis Calibration DP DP default itk 16 14 DP uCal sec 0 500 1000 1500 2000 2500 3000 3500 4000 Time sec uCal sec button Origin will ask for the DP check file Select the data file just created and click Open The computer will think for a few moments If any of the pulses are out of specifications a pop up will inform you It will ask you to save Origin will show four graphs The upper left graph holds the raw data The lower two graphs show the energy and power of each pulse The upper right graph displays the percent error for the energy and power of each pulse Right click at the upper right portion of the graph and select Go To Window from the menu Check the sizes of the errors If any of the errors is greater than 1 please contact MicroCal 3 6 Temperature Calibration Check It is recommended that the ITC temperature calibration be checked approximately once every 12 months The measured temperatures should be within 0 2 degrees of the set temperatures in the 11C200 software This procedure requires a Control Company digital meter and submersible probe or equivalent
6. general categories of troubleshooting for the 1TC gt 00 and its operation The most extreme category is when a system 1s not working at all Problems that prevent users from operating the 1T C gt 20 require immediate consultation with a MicroCal technician Customers should not attempt to repair the 1TC20 hardware or software unless instructed to do so by a MicroCal service representative The second and less extreme general category of a problem is when an 1T Co instrument is functioning but is not operating within its normal performance specifications Large baseline drifting non repeatable control peaks water water and or an increase in short term noise level are examples of performance problems These problems may be corrected by the operator in most cases For these types of performance issues it is recommended that customers carry out the following minimum diagnostic steps prior to contacting MicroCal for service 1 Thoroughly clean the cells Do not assume they are clean build up or unexpected sample residue will cause problems As a minimum use the provided Cell Cleaning Apparatus to pass at least 50 ml of 10 15 detergent in water solution followed by 50 ml of water through the sample cell Discretion may call for a more rigorous cleaning procedure 2 Using aclean Hamilton syringe refill both the reference and sample cells with filtered degassed water Thoroughly clean the titration syringe or use a different syringe Load the syri
7. shorter equilibration times Also high temperature thermostatting during cell cleaning can improve the effects of the cleaning Use the arrow buttons to raise or lower the temperature or click in the textbox and type in a new number Click Set Jacket Temp to set the temperature The Pulse Control section allows for manually administering a DP calibration pulse While this is not the most thorough method of checking the y axis calibration it is the quickest method Pulses may be applied any time the DP signal is equilibrated and the resulting deflection used as a crude calibration assessment If Origin for real time plotting is enabled Origin will calculate the error If this is greater than 1 please see Section 3 5 for a more thorough check of the DP calibration The Pipette Control panel at the center of this tab provides the controls for loading and cleaning the syringe The Open Port button moves the plunger tip to above the fill port in the side of the syringe Close Port moves the plunger tip down so that it blocks the fill port Purge Refill pushes the pipette tip all the way down and back up again dislodging bubbles on the sides of the syringe The Pipette Maintenance section provides the software controls used for changing the pipette tip Please see Section 3 4 for details on performing this maintenance The Run Properties bar at the bottom of this tab lets the user choose output options Postrun analysis will be carried out only if Auto
8. 49034 0 752929 Max Safe Temp or JE ATP Read 8 O DP FB Mode 4 D Slope Slope DP Passive FB 06687 ITC Injector Coefficients pr Low Gn FE eS Steps Per Inch 256000 DP Mid En FE 29862 Max Safe Postition 260972 steps DF High Gn FB me Syringe Constant a ulin Silene Olite ca Mani ner T2 Set Point 0033503 0 027142 APM 1 RPM 2 RPM 3 RPM 4 ATP Output 18 6 ATP Time Shift EG sec Start in Advanced Mode tells the software to open with the Advanced Experimental Design tab selected by default The ITC Equilibration Options affect the equilibration before a run starts and are generally only used for troubleshooting When the Fast Equil option 1s disabled the system will go through an additional prerun state a non stirring equilibration period before stirring 1s started This can be useful when stirring related noise problems are suspected When the Auto Mode option is selected the ITC experiment will proceed through the equilibration states Pre stirring and Final Baseline and complete the experiment without any further interaction from the user With this option disabled the prerun equilibration will not progress from one state to another until the user double clicks on the DP data box E ITC200 Default User ITC Options System Contact Info About Experimental Dy I lane C mba rm The Help gt Contact Info menu option provides c
9. Cal sec The Extended Data Mode button will tell the software to save all data the 1T Czo0 instrument produces to the data file This provides MicroCal engineers with extra data for troubleshooting The Export Current Data button will bring up a save as window to prompt the user for a filename and then save whatever 1s currently displayed in the real time window to that file By default it will be a dat file 2 7 Menu Options The main drop down menus at the top of the 1TC200 software will provide access to some of the less frequently used functions of the application The System gt Quit Program menu option prompts the user to confirm that they want to terminate the application Responding with Yes will terminate the program and any run in progress All ITC run data will be saved to disk Approximately a minute or so after program termination the power to the 1T C gt 99 will also be shut down Power to the ITC will not be restored until the application is run again T ITC200 Default meee TE Options Quit Program The ITC gt Print Save As Text Run Parameters menu option allows users to print or save to a file the run parameters that are currently loaded in the iTC20 software While run parameters are always included as part of the ITC data file header the Print Save As Text Run Parameters menu option provides the user with a nicely formatted listing of run parameters ideal for data presentation or general re
10. al Design Advanced Experimental Design Real Time Plot Setup C value within normal range 1 Experimental Mode Results Minimum Protein r 2 Starting Yalues Cell uti Change 130 Help Syringe pb Change 1450 Help C Change Bo Help Please enter H value and press Enter Expected H 1 Molez Mole Estimated Kd po00005 MM Help Delta H 17 kcals Mole C Yalue Legend Optimal Temperature 30 Degrees C 5 500 Data File Name i O default itc 500 1000 Update Experimental Curve F Extremely Outside of Optimal Options E Use Ka 17M Outside of Optimal Range 1 5 Po NDH DH oo ne Ado SE Normalized Molar Ratio When the software is first started by default the Experimental Design tab is selected this contains the simple run controls Experimental Mode can be Highest Quality or Minimum Protein Highest Quality uses 20 injections and a c value of 100 These parameters should produce data that 1s clear and easier to fit Minimum Protein uses fewer injections 10 and a c value of 5 The result of these parameters will be the use of the least amount of sample necessary for a successful titration The expected n Ka and AH and the desired run temperature will allow the software to calculate the recommended concentrations for the cell and syringe and set the run parameters based on mode chosen If the user is unsure of the Ka for their system clicking the Help butto
11. astic tubing from the upper tube of the cell cleaning apparatus into a beaker of 50 100 ml of detergent cleaning solution Connect the tubing to the top of the waste flask Make sure that the other connection on the flask is connected to the ThermoVac and that the ThermoVac cover is seated Turn on the ThermoVac vacuum pump The vacuum will pull the detergent solution from the beaker through the cell and into the waste flask Do not allow the waste flask to become full enough for liquid to be sucked into the ThermoVac s vacuum pump as this may cause damage Once sufficient detergent solution has passed through the cell remove the plastic tubing from the solution and placed into another beaker containing 100 ml or more of water for rinsing After rinsing with water remove the plastic tubing from the rinse water and allow time for the vacuum to drain the fluid out of the plastic tubing then remove the cleaning apparatus from the cell and remove the remaining water from the cell using a Hamilton syringe If this cleaning method is insufficient fill the cell with 5 LiquiNox and set the instrument to thermostat at 70 C for several hours Be careful to cool the cells before removing the LiquiNox as hot liquid may shatter the loading syringe Syringe Cleaning Conceptually cleaning the syringe is similar to using the ThermoVac to clean the sample cell the vacuum pulls liquid from a beaker through the syringe and into the waste beaker in t
12. ations or modifications must only be carried out by specialist personnel or with explicit directions from a MicroCal technician Removal or modification of any cover or component could result in an unsafe or easily damaged instrument The MicroCal service department will be happy to answer any questions and provide parts and service when necessary 1 A solution can become an electrical conductor when in contact with electricity Use caution when using solutions near the instrument If any liquid is spilled on or around the instrument unplug the instrument immediately and wipe it up Be careful not to overflow the small reservoirs around the cell access tubes If there is any possibility that liquid may have leaked into the instrument case contact MicroCal immediately Do not plug the instrument into any electrical outlet until the problem is resolved 1 The operator should always follow proper laboratory procedures in handling and disposing of volatile or hazardous solutions i All solutions in the cells must be cooled down below 40 C before removal Any higher temperature may cause the syringe to break and will increase the dangers of most hazardous solutions 1 Never allow liquid in the cells to freeze The expansion of the liquid can distort the cells and rupture the most critical sensor causing irreparable damage The iTC instrument should always be moved in its normal operating orientation Other orientations will su
13. bject delicate sensors inside the instrument to stress The 1T C20 cells are constructed out of Hastalloy Strong acids should be avoided Please refer to the accompanying material analysis booklet for further information This instrument is not designed to the Medical Devices Directive 93 42 EEC and should not be used for medical purposes and or in the diagnosis of patients Instrument Specifications i Performance Specifications Operating Temperature Range 2 80 C Response Time 10 seconds Cell Design 200uL coin shaped Titration Syringe 40uL Maximum Usable Volume 35uL Smallest Injection Size 0 1uL Stirring Rate 300 1500 RPM Physical Specifications Cell Material Hastelloy Alloy C 276 Dimensions Calorimeter 6 x 13 x 10 Controller 15 x 16 x74 Monitor 17 x 16x 5 Weight pounds Calorimeter 16 Controller 20 Monitor 9 5 Electrical Specifications All electrical specifications are for the calorimeter only Electrical Ratings Voltage 110 240 Volts Frequency 50 60 Hz Power 70 Watts Fuses 2 3 15A 250V Time Delay Output Secondary Data Connection Only Protective Earth Terminals Internal external marked Mode of Operation Continuous Classification Class I Atmospheric Specifications Operating Temperature 10 40 C Humidity 0 70 RH non condensing Atmospheric Pressure 700 1060 HPa Storage no liquid in cells Temperature 40 70 C no liquid in c
14. cceeding several points and must be removed for a better fit Many users will make a smaller first injection to minimize sample usage on this first injection 5 Click on the One Set of Sites button at the top of the Model Fitting section A new window will pop up and a red curve will appear in the graph window 6 Click on the 100 Iter 100 iterations button until the Chi squared is as low as it will go The red curve will come closer and closer to the curve formed by the delta H points The dialog box will indicate when the Chi squared is no longer being reduced This means that the fit is as good as the computer can make it Example dialog box text Press Esc key to stop fitting iterations 1 Levenberg Marquardt Successfully progressed 4 rounds Reduced Chi sqr 3510 72797 Total 4 rounds in this session 2 Levenberg Marquardt Successfully progressed 1 rounds Reduced Chi sqr 3510 69257 Total 5 rounds in this session 3 Levenberg Marquardt Successfully progressed 1 rounds Reduced Chi sqr 3510 69257 Total 6 rounds in this session 4 Levenberg Marquardt Chi sqr is not reduced Reduced Chi sqr 3510 69257 Total 6 rounds in this session Click Done A textbox will appear on the graph window 7 Select Origin menu ITC
15. cord keeping ITC200 Default User stem Pe Options Help I Stark ITC Calibration Run Temperature Check Ir User Ser Axis Check The Pipette Tools menu option opens a popup window for adjusting syringe calibrations The Start ITC Calibration Run menu item 1s used to check the accuracy of the instrument s calibrations See sections 3 6 and 3 7 for more information The Options menu allows for several user settings Auto postrun data analysis tells the computer to automatically analyze the data after each run The exact procedures are controlled from the bottom of the Instrument Controls tab ITC 700 Default User stem ITC Bend Auto postrun data analysis ig Heating Tc System Coefficients Start in Advanced Mode Je TE Fe Experir ITC Equilibration Options ow Fast Equil ss OSEN y Auto Mode System Coefficients opens a popup window with the most critical of the software calibration constants This is often the easiest place to find the instrument serial number which is in the upper left corner None of these numbers should be changed without direct instruction from a MicroCal technician T System Coefficients VRITC 2 07 030 Cell Parameters 1 0 Channel Coefficients Cell olume TES Slope Offset Ref Cal Htr es JacketTRead 2935131 083274 Samp Cal Htr 169000 Delal Read 0 8333333 Min Safe Temp 1 C Shield T Read 29
16. e Save Run File Advanced Experimental Design Instrument Controls Real Time Plot TO Pipette Accessor Station Thermostat Control _ Pipette Controls Set Point C in Open Fill Port Close Fill Port Set Jacket Temp P E Fill Pulse Control _ Purge gt ReFi Set Up Pulse Size Distance in Up EE ME bi Dn Duration 300 pet Maintenance _ 1 Remove Old Tip 2 Install New Tip 3 New Tip Installed Auto Postrun Data Analysis Properties Run Type Experiment Control Excel File Name 0 laa 0 Control Subtract Options Do not Subtract Control Run File Hame The Instrument Controls tab contains the controls for direct operation of the instrument At the top of the window the user can Start Run using whatever parameters are currently present in the Experimental Design or Advanced Experimental Design tabs Before clicking this button it is wise to check that all parameters are correct and that a valid unique data file name has been entered The software will double check with the user before allowing any files to be overwritten The Stop button which is available only during a run will abort the run immediately The Thermostat Control section allows for setting of the thermostat temperature which will be maintained during the 1TC gt 00 thermostating idle state Pre thermostatting the 1TC20 and samples at the run temperature will result in
17. e The syringe will start to rotate The start of the stirring will cause the DP signal will deflect downward and then will increase but will stabilize at a level slightly lower than the non stirring baseline The instrument will stir continuously for the remaining portion of the run Once the DP signal has stabilized after the start of the stirring the instrument will enter the run state The run commences with an initial delay to provide baseline data prior to the first injection Then the injections will be executed according to the injection schedule The instrument responds to an exothermic reaction by decreasing the feedback heat provided to the sample cell That 1s heat added to the sample cell by an exothermic reaction will result in a downward deflection in the baseline DP signal This downward deflection is proportional to the energy not needed to maintain the temperature equilibrium between the sample and reference cells An endothermic reaction will require more heat in the feedback loop to maintain the same temperature resulting in a positive deflection The integrated area of this deflection is referenced to the baseline before the injection and after the reaction has completed and a curve is fitted to the points generated In the single injection method the shape of the peak from one injection is used to determine the characteristic curve 2 5 Real Time Plot e ITC200 Default User System ITC Options Help Thermostatting ITC 30
18. e dialog box which allows saving of the currently displayed run parameters Once the parameters have been saved they may be reloaded and reused in the future by selecting Load Run File as described above Display Run Parameters displays the current run parameters for the run in progress This button is available only when the 1TC201s in a non idle state Update Run Parameters updates the current run parameters for the run in progress When the ITC is in a non 1dle state this button must be clicked for the run parameter changes to take effect This button is available only when the 1TC200 is in a non idle state Note Only run parameters that have not yet been applied on the run in progress may be updated all others will be ignored 1 e after the stirring has commenced for an experiment changes to the experimental stir speed will be ignored The Total Injections 500 is the maximum sets the number of injections for the titration ITC experiment The multiple injection method requires a minimum of 10 15 injections There are no negative consequences except the time spent to titrating past the point where all sites are saturated and these last few injections can be used for control heat information The single injection method uses one single longer injection The Cell Temperature box is used to set the desired run temperature for the experiment Most runs are performed between 25 C room temperature and 37 C human body temperature althou
19. e provided with your instrument to make a diagonal cut on the side of the white Teflon tip image 1 and use the small tipped tweezers to pull the tip off image 2 Click the Install New Tip button to position the tip Use the tip pusher tool with the new tip in the slot hole side up and firmly press the new tip into place image 3 5 Rotate the tool to ensure that the tip is firmly seated Remove the tip pusher tool and inspect the tip to be sure it is seated properly Reinstall the syringe as described above to finish 3 5 Y axis Calibration Check It is recommended that the y axis calibration be checked every few months to ensure accurate data acquisition The automatic calibration check routine will send a series of pulses to the cell heaters dissipating a known power The offset in the DP as a result of this power is analyzed in comparison to the correct DP offset Make sure the cells are clean and fill both cells with degassed distilled water Load the titration syringe with water and insert it into the sample cell It is recommended that Origin for real time data be enabled Ni VPViewer2000 System ITC Help Print Save As Test Hun Parameters Start ITC Calibration Aun w ITC Y Axis Calibration File DP Calibration Parameters Experimental Temp 30 Data File Name defaultitk Humber of Pulses le Reference Power in Filter Period PE Stirring Speed 300 Feedback Mode Gain Hone CC Low tf High Cal
20. ells Humidity 10 90 Atmospheric Pressure 500 1060 HPa Section 1 Introduction to the iT Czo This section provides information about isothermal titration calorimetry and the basic parts and operation of the Isothermal Titration Calorimeter ITC instrument and software At the end 1t includes several suggested tutorial experiments 1 1 The Basics of ITC The 1TC20 Isothermal Titration Calorimeter 200uL cell unit directly measures heat evolved or absorbed in liquid samples as a result of mixing precise amounts of reactants A spinning syringe 1s utilized for injecting and mixing of reactants Spin rates are user selectable the usual range is 500 to 1000 RPM The normal temperature operating range 1s 1 C to 80 C Wetted cell surfaces are Tantalum or Hastalloy which are resistant to most solutions however strong acids and strong bases should be avoided Sample and reference cells are accessible for filling and cleaning through the top of the unit The sample cell is on the left as one faces the front of the unit A pair of identical coin shaped cells is enclosed within two shields the inner shield is referred to as the jacket Access stems travel from the top exterior of the instrument to the cells Both the coin shaped cells and the access stems are completely filled with liquid during operation This requires approximately 250 uL per cell even though the working volume of the cell is only 200 uL Temperatu
21. ental Design tab was used these values will already be filled in These concentration values are stored in the data file header and used for ITC data analysis These values are used only during data analysis and an entry is not required to perform an experiment Select the desired stirring rate from the drop down list 1000 RPM is recommended for most ITC experiments If the solution in the sample cell contains suspended particles e g agarose beads then faster stirring may be necessary In the Feedback Mode Gain section the three available modes are none low or high High is recommended for most ITC experiments The feedback modes can be described as active low or high or passive none High gain will provide the fastest response time while passive mode none will provide the highest sensitivity Almost all ITC reactions will require using the high gain feedback mode Monitoring of long slow thermal process in the iTC2oo i e kinetics metabolic rates might benefit from using the passive or low gain feedback modes The Data File Comments box allows the user to enter comments about the experiment to be saved in the header of the data file for future reference The simulation plot in this window is generated from the Experimental Design tab Clicking Update Experimental Curve pulls the information from that tab to the current one In addition the user may enter the K4 and dH here and change the plot from this tab The Injection Paramet
22. er I se oe Y Axis Scale __ Extended Data Mode El So ada Export Current Data pCal sec Thermostatting ITC 30 C Time Left fs lt gt pat The Setup tab allows customization for each user This is often most helpful when different users have different default data file paths so that each user s files are stored in a different place in the computer The Data File Path box indicates the path excluding file name where 1TC20o data files will be saved The path should be located on a local hard drive and not on any floppy or CD ROM drive This setting may only be changed while the 1TC20 1s in the idle state thermostatting To change the current data file path double click on the existing path text and a dialog box will open allowing the user to change the path The desired data directory must already exist in order to select it from the dialog box In order to be read into Origin for data analysis the path cannot contain any spaces The Setup File Path box indicates the path excluding file name where 1T C200 setup run files will be saved and loaded The specified path should be located on a local hard drive and not on any floppy or CD ROM drives To change the current setup file path double click on the existing path text and a dialog box will open allowing you to change the path The desired setup directory must already exist in order to select it from the dialog box The Init Setup File box specifies the initial setup f
23. ers group allows the user to define the volume duration spacing between injections and the filter period for each injection At the bottom of this group 1s a summary table that lists these parameters ordered by injection number The user may select an injection to edit by clicking on a row to highlight it the parameters for this injection can be entered into the text boxes see below Then depending on the Edit Mode selected the user may edit the text boxes to effect all injections All Same only the currently highlighted injection Unique or the currently highlighted injection and all the following injections Apply To Rest In the Volume box the user may enter the volume of titrant in uL to be injected from the pipette into the sample cell for the selected injection s In the Duration sec box the user may enter the time in seconds that the instrument will take to inject the titrant into the sample cell The 1TC20 software will generally default this value to be twice the number entered in the Volume text box For Single Injection Method SIM experiments the standard duration is ten times the volume In the Spacing sec box the user may enter the time in seconds between the beginning of the selected injection and the beginning of the next injection or end of the run This spacing must allow enough time between the injections to allow the DT signal to return to the baseline after an injection peak deflection Typical values
24. for this parameter range from 90 180 seconds depending on the size of the peak and the kinetics of the reaction For Single Injection Method the spacing should be 90 180 seconds greater than the duration 1 e volume 20 ul duration 200 seconds spacing 300 seconds The Filter Period sec is the time period in seconds in which the data channel conversions are averaged to produce a single data point for plotting and saving For fast reactions a filter period of 5 seconds is sufficient to obtain enough data points for representation of the peak for accurate integration of the area For monitoring of very long slow thermal processes the filter period may be increased accordingly to avoid accumulation of excess data points Although there may be a practical limit for a data set size only available disk space would limit the number of data points that the 1TC20 software is able to collect and save Calculating Concentrations The easiest way to determine a good starting concentration for a system 1s to use the Experimental Design tab of the 1TC20 software see section 2 2 This section provides an alternative method for the slightly more advanced user To approximate the sample concentration needed in the sample cell estimate the Ka for the system and multiply by 10 With very tight binders the cell concentration should be on the low side of this value for weak binders the concentration should be on the high side of this value Mul
25. gh the instrument s operating range is 2 C to 80 C Throughout an ITC experiment a small constant amount of power equal to the Reference Power entered is continuously supplied to the offset heater of the reference cell This causes the DP feedback system to become positive to supply compensating power to the sample cell that will equilibrate the temperatures During an experiment the DP baseline will equilibrate near the value entered in the Reference Power box The reference power setting 1s often referred to as the baseline setting The best choice for the reference power setting will be determined by the anticipated size and direction of the titration peaks Large exotherms will require a large reference power setting ca 5 UCal sec and large endotherms will require a very small reference power setting ca 0 5 uCal sec When working with an unknown sample a reference power of 2 3 uCal sec is best The Initial Delay refers to the time in seconds after the instrument has started a run and before the first injection The standard parameter 1s 60 seconds this is necessary to establish a baseline prior to the first injection The Syringe Concentration and Cell Concentration boxes are a place to enter the experimental concentrations The Experimental Design tab will make suggestions of the proper concentrations 1f desired see section 2 2 or refer to Calculating Concentrations later in this section for more information If the Experim
26. gt Final Figure A new layout will be created with the raw data in the top graph and the delta H data in the bottom graph Section 3 Instrument Maintenance This section provides the user with information on the proper maintenance of the instrument to ensure proper function The tools are pictured and labeled below Soft Grip Tweezers Small Tipped Tweezers Pipette Tip Cleaning Wire 3 1 Cleaning the Cells and Syringe Cell Cleaning The 1T C20 uses fixed in place cells in order to provide maximum sensitivity and stability These cells must be cleaned routinely to maintain the high performance of the instrument Dirty cells will contribute greatly to cell filling problems accuracy and repeatability problems and possibly misinterpretation of data Inadequate cleaning is the cause of many problems experienced with the 1TC 0 The reference cell generally requires no special cleaning rinsing and refilling every week or so 1s sufficient The mildest method of cleaning the sample cell 1s simply to rinse the cell with buffer several times before loading the sample This should be done whenever the necessary buffer is available Every day or two or after any sample that precipitates the sample cell should be cleaned with detergent using the Thermovac Insert the long needle of the cell cleaning apparatus into the sample cell and push down carefully until the o ring has sealed as shown below Immerse the end of the pl
27. he ThermoVac chamber With the syringe in the sample filling hole attach the filling tubing You will need to line up the hole in the pipette with the hole in the side of the syringe Spin the syringe assembly to align the syringe fill port with the threaded hole on the side of the pipette Screw on the end of the tubing see image below Attach the end of the tubing to the port on top of the waste flask and make sure that the waste flask is attached to the ThermoVac Move the pipette so that it is in the cleaning hole furthest from the cell Push down gently until it seals in place Turn on the ThermoVac s vacuum Hold the end of the tubing in a beaker of 5 Liqui Nox for several seconds Make sure that liquid is being pulled into the syringe and through the tubing to the ThermoVac Hold the end of the tubing in a beaker of laboratory grade methanol for several seconds and leave the end open to the air for several minutes to dry the syringe Once it 1s dry turn off the vacuum and disconnect the tubing at the ThermoVac Move the pipette to the sample filling hole to fill the syringe If clogging of the syringe 1s suspected it should be cleaned by running the thin wire through it This must be done very carefully First remove the syringe from the pipette see section 3 2 As always when handling the syringe be careful not to bend the needle The wire should always be inserted through the glass first both to ensure that the clog is fully re
28. ibration Power uCalfsec 1 Pulse Duration sec 300 Pulse Spacing sec 600 Start Run Cancel To begin the y axis calibration check procedure select iTC20 software menu ITC gt Start ITC Calibration Run gt Y Axis Check Once the menu has been selected the Calibration Pulse Setup Window will appear This window allows the calibration pulses to be modified Individual pulse parameters are entered by first selecting a pulse or multiple pulses then entering the desired parameter value into the pulse parameter boxes Calibration Power Pulse Duration and Pulse spacing Users are encouraged to simply use the default y axis calibration parameters After the run and pulse parameters are entered clicking on the Start Run button will start the run The ITC will equilibrate in the same manner as it would during a titration experiment If creating customized calibration parameters users must be aware of the DP range limits when setting reference power and pulse sizes The reference power must be high enough to allow all pulses without hitting saturation and if a pulse size is too small it can show abnormally high error After the final equilibration phase has completed the initial delay will begin and the pulses will be applied as entered As each pulse completes Origin will analyze the pulse region and determine the deflection of the baseline as well as the energy area of the pulse The requested power and energy will also be
29. ile which will be loaded into the experimental control window when it 1s first opened Specifying the initial setup file as LastRun 1 inj will result in the last run parameters that were executed to be loaded at startup The Cells Boot Temperature box sets the temperature that the instrument will heat or cool to and then thermostat at when the 1TC20 instrument 1s powered up To save equilibration time set this temperature to the same as the common experimental run temperature The Analog Input Range selector determines the maximum range of measurable DC voltages for all iTC20 data channels The default range of 10 volts is adequate for almost all ITC applications and provides for the greatest sensitivity Please consult a MicroCal engineer prior to making any changes The Save Add Erase User buttons provide a convenient way of maintaining unique startup parameters and placing data files in different folders for multiple users To enable additional users select the Add User button and enter a user name in the text box and click OK Save User will save the current settings for the current user Erase User will remove a user The Y axis group allows for changing of the current y axis DP units Available y axis units are mCal min wCal sec and uWatts By default y axis units will be displayed in uCal sec Changing y axis units will only change the power units for data displayed in real time All data files will be saved with the default units of u
30. jection DH or the heat normalized to the molar ratio NDH 2 3 Advanced Experimental Design The advanced experimental design tab may be used in addition to the Experimental Design tab for operating the 1TC gt 0 instrument Below is a description and suggested values for each of the run parameters displayed in this tab dt ITC200 Default User System ITC Options Help Thermostatting ITC 30 C Time Left Fo I Load Run File Save Run File Experimental Design Advanced Experimental Design Instrument Controls Real Time Plot Experimental Parameters t_ Injection Parameters Total Injections Volume pl Edit Mode All Same lt gt Unique lt gt Apply To Rest 20 40 1 180 5 Data File Mame default ita Feedback Mode Gain oo 20 4o 180 lt gt None lt gt Low High A 40 180 Data File Comments Kd 0 000005 dH 17 MDH a 1 2 3 Normalized Molar Ratio The top bar in this tab contains several buttons for opening saving and viewing run parameter files The Load Run File button will display the file open dialog box in which the user can select a file to load into the Advanced Experimental Design tab Run parameters may be loaded from one of two types of files a previous experiment s data file itc the run parameters are extracted from the data file header or a setup file inj which was previously saved using the Save Run File button Save Run File displays the file sav
31. ls Experimental Mode can be Highest Quality or Minimum Protein The expected n Ka and Delta H and the desired run temperature will allow the software to calculate the recommended concentrations for the cell and syringe and set the run parameters The Advanced Experimental Design tab contains more direct controls for the more advanced user This tab should be very familiar to users of the VP line of instruments The Instrument Controls tab allows the user to name the output files choose post run analysis options and start and stop the run The Real Time Plot tab shows the data currently being generated The Setup tab contains various options and preferences Optional Origin The copy of Origin that is opened by 1TC200 software if desired is for real time data display For analysis the user should open a separate copy At the top of the graph screen the current status of the instrument is shown Below that the file name that the software is currently saving to is displayed The graph displays DP in wCal sec verses time in seconds The Main Display box in the upper left corner shows the temperature of the cells current DP and DT The lower left corner contains the graph view options Rescale To Show All resizes the window to fit whatever data is currently displayed in it Auto View 1 and Auto View 2 set the window to a certain scale with the latest data point centered vertically and Saved View 1 and Saved View 2 set the Y axis to a specific ra
32. moved from the syringe and to decrease the likelihood of bending the syringe It may be difficult to insert the end of the wire from the glass bore into the metal needle good light and a magnifying glass will help Continue to insert the wire until 1t emerges from the tip of the needle see image below Carefully pull the wire back through the needle and glass bore NA a ttt NN a Y NE MN Gamo sg NOM Bear AN Y yo wS i RA cae 3 2 Changing Injection Syringes Clean and dry the syringe before removal Remove sample tube from loading hole and disconnect the cleaning apparatus Be very careful whenever the syringe 1s not secured in the pipette as the needle bends extremely easily See images 1 7 below for illustrated removal Slide the pipette to the cleaning position and insert it firmly into the hole Turn the inner metal syringe holder to the right several full rotations This will unscrew the metal ring that holds the syringe in the pipette One the ring is loose move the pipette to the empty loading hole Use the manual pipette controls to lower the pipette tip about 0 3 inches Without the bottom ring the syringe will move down with the pipette tip Enough of the syringe bore should now be exposed to hold it firmly and pull straight down pulling the syringe out of the pipette and down through the filling hole The soft grip tweezers can be used to help grip the syringe without damaging it Carefully put the syringe in a
33. n causes the software to prompt for the type of compound in cell and syringe It will then make a guess as to the Ka The user will still be required to choose values for AH and n Click the Update Experimental Curve button to calculate the results The simulation window will update with a rough graph and the Results column at the right of the screen will have values for the cell and syringe concentrations The calculated C value is listed below its background is color coded This value is equal to 5 in Minimum Protein mode 100 in Highest Quality mode and will vary if the user chooses to change cell concentration manually The c value predicts the shape or sigmoidicity of the curve Optimal values for C are between 5 and 500 green values between I and 5 and 500 and 1000 should work but may not give the best result yellow C values less than 1 or greater than 1000 will probably not yield usable data red The user may adjust the two experimental concentrations by using the change buttons beside each concentration box Any warnings such as heats too high for the instrument to measure will appear in the status bar near the top of the screen It is highly recommended that the users look carefully at the projected curve and make sure that the shape and rough values are reasonable before proceeding A pair of options at the bottom of this tab allows users to work in Ka or Ka and to choose whether to view the simulation plot using raw heat per in
34. ng follow the same procedure as for the sample cell If there is a saved run file for the current experiment load the run parameters by clicking the Load Run File button at the top left of the Advanced Experimental Design tab in the 1TC20 software and select the run file There are standard run files for most of the tutorial experiments If no run file exists enter parameters in the appropriate boxes in the Experimental Design tab see sections 1 3 and 2 2 for more information Once the parameters have been entered click the Start button at the upper left corner of the Instrument Controls tab of the control software Be sure to insert the syringe in to the sample cell before starting the experiment The instrument will seek experimental temperature equilibrate to that temperature start the titration syringe stirring wait until the DP signal is steady and then start performing injections The raw data will appear in the Real Time Plot tab Once a run has finished the syringe and sample cell should be cleaned as soon as possible See Section 3 1 1 5 Tutorial Experiments The next two tutorial experiments are recommended for every new user before beginning analysis of their own samples They are designed to provide standard results which check that the instrument is working well and the user is comfortable with the procedures before risking precious sample Water Water Tutorial The first tutorial involves titrating water into water
35. nge All four of these views can be edited through the Edit ranges button 1 4 Basics of Performing a Run In order to perform a basic ITC titration experiment the user must load the sample cell and the syringe enter the desired parameters into the control software and click Start The reference cell should be filled with water or buffer and may be left for several days Loading the Syringe To load the titration syringe place a microcentrifuge tube in the tube holder Be sure to push the tube to the bottom of the holder with the lid fitting into the slot provided see image below Be careful not to leave any part of the tube in the path of the syringe needle to prevent damage Disconnect the filling tube from the ThermoVac unit and connect the threaded end to the pipette fill port if necessary it should still be in place after cleaning and drying of the syringe Insert the tip of the 1 mL plastic syringe into the end of the filling tubing In the software under the Instrument Controls tab click the Open Port button to make sure that the pipette fill port is open Slide the pipette up across and down into the middle slot so that the tip of the syringe rests in the microcentrifuge tube Gently pull up on the plunger until the sample has filled the titration syringe and is just overflowing into the tubing Click the Close Port button to close the fill port Pull the plunger up approximately another 0 1mL to remove the excess liq
36. nge with water 3 Go to the Setup window within the 1TC20 software and activate the extended data mode While the software 1s in the extended data mode the ITC data files will contain all available information produced by the 1TC290 This additional information will often help the MicroCal technician diagnose problems 4 Carry out a minimum of 20 luL injections of water into water If after completion of the 4 steps listed above the ITC performance is not corrected please contact the MicroCal service department for help The water run in extended data mode should be provided to the MicroCal service technician for evaluation Following the evaluation a representative from the service department will contact you with comments and recommendations MicroCal LLC Contact Information Primary Contact Phone 413 586 7720 Toll Free Phone Number 800 633 3115 Fax 413 586 0149 European Office 44 1908 576330 European Fax 44 1908 576339 Web Site www microcal com Service E mail support microcal com Customer Service E mail customerservice microcal com Please forward corrections or suggestions concerning this manual to info microcal com Origin is a registered trademark of Origin Lab Northampton MA MicroCal is a registered trademark of MicroCal LLC Northampton MA Windows is a registered trademark of Microsoft Corporation Symbols Symbols used in this manual Warns the user of possible damage to the unit draw
37. ontact information for MicroCal support The About menu option provides specific details about this version of software 2 8 Origin Real Time Display This section describes the functionality of the optional copy of Origin for real time display When the software is opened it will open the Origin project window VPITCPLOT OPJ for real time data display This project of Origin is dedicated to data display only and should not be used for data analysis Users should open a separate copy of Origin for ITC to perform data analysis Pictured below is the main Origin window for ITC2o data display f Origin 7 C Origin70WPITCPLOT OPJ Read Only Plot1 MEG File Edit View Graph Data Analysis Tools Format Window Help fel x b fem elol Aaa bo 6 1 EO Te _ Current Status Thermostatting ITC 30 C Main Display JTemp1 DP DeltaT1 JT Temp C 30 2 ji as 10000 10 10 ITC Cell Status DP pCal sec 4 096 DT C 0 003 8 8 5000 ITC Numeric Display o D 6 6 Display Mode 2 o A Rescale To Show All O 4 4 r DP Scale Controls Buttons for ITC Auto View 1 5000 Data Display Auto View 2 Saved View 1 Saved View 2 10000 0 0 j Time sec i Edit ranges We izeia EA qa YPITCPLOT OPJ Name Type 1 Datal DP 1 0 Ploti Radian The ITC cell status the ITC2oo numeric display and the buttons for ITC20o data tools as indicated above have been added for
38. orrect order First boot up the computer and log in to Windows Once Windows has started power the 1T C20 by operating the switch at the rear of the unit After several seconds open the iTC2o software If the option is selected a real time copy of Origin will open automatically as well as the 1TC200 control software e ITC200 Default User System ITC Options Help Thermostatting ITC 30 C Time Left Fs u Load Run File Save Run File Experimental Design Advanced Experimental Design Real Time Plot 0 Setup O 1 Experimental Mode Results Please chooze Cell uh _Chanae fo Help Syringe uti Change oo Help E Change oo Help Expected H oo Moles M ole Estimated Kd M Help Delta H kcale Mole C Yalue Legend Temperature 20 Degrees E Melis Data File Name O default tc FOO 1000 Update Expenmental Curve Extremely Outside of Optimal Range 1 1000 Outside of Optimal Range 1 5 Options E Use Ka 14M Pat MDH 0 4 0 6 Time 3 The line just below the menus reads System Initiation Please Wait which 1s the current status of the instrument After a few seconds the system will begin heating or cooling to the preset temperature To the right of that the Time Left box during a run will show the time left until the end of the run When the software is first started the Experimental Design tab is selected this contains the simple run contro
39. place where it will not roll or be damaged See images 7 12 for illustrated syringe replacement Gently push the syringe up through the filling hole and into the pipette It will come to a stop with about 4mm of syringe glass exposed below the metal Carefully push up on the glass while spinning the metal syringe holder slowly Once the notch in the syringe aligns with the notch in the holder the syringe will slide up approximately another 2mm Move the pipette to the cleaning hole and turn the inner metal syringe holder to the left until the bottom ring 1s secure 3 3 Removing Pipette The pipette is held by the arm and connected to the tower by two small cables Detach the two cables from the top of the tower First pull up on the tiny tabs on each side of the locking pieces The cables should slide out of their sockets Loosen the screw on the side of the arm to remove the pipette To reinsert the pipette place the pipette in the socket on the arm and tighten the screw Insert the cables into their slots and press the locking pieces back down to hold them The white cable goes in the slot to the rear of the instrument and the orange brown cable goes into the front slot 3 4 Changing Pipette Tips See the instructions above to remove the syringe from the pipette The pipette should remain connected to the tower In the Instrument Controls tab click the Remove Old Tip button to position the tip Use the blade of the X acto knif
40. postrun data analysis is selected under the Options menu Run Type tells the computer whether this run is an experimental run or a control run The textbox to the right of this allows the user to enter an excel spreadsheet file name In the bottom left textbox are the options for control heat subtraction Do not Subtract tells the computer to do nothing Subtract Saturation Points will cause the automated data analysis to shift the delta H data so that the final blank heats are zero Subtract Straight Line will fit a straight line to the data and subtract it Subtract Control File will subtract the file named in the Control Run File Name box Clicking the browse button will open a pop up in which the user can browse to the appropriate file The file name can also be typed in directly States of Operation The 1TC200 IS a state driven instrument The current state of the ITC cell status is displayed in both the control software and Origin if it is enabled There are five unique states that comprise an ITC run thermostatting idle seeking temperature prerun thermostat final baseline equilibration and running A description of each of these five states can be found below The thermostatting idle state is what is in effect when no run is being performed The 1TC200 will achieve and maintain the currently defined thermostat temperature It will remain in its idle state until the operator starts a run by clicking the Start Run button When a run i
41. re differences between the reference cell and the sample cell are measured calibrated to power units and displayed to the user as well as saved to disk The data channel is referred to as the DP signal or the differential power between the reference cell and the sample cell This signal is sometimes thought of as the feedback power used to maintain temperature equilibrium Calibration of this signal 1s obtained electrically by administering a known quantity of power through a resistive heater element located on the cell In a typical experiment the syringe containing a ligand is titrated injected into the cell containing a solution of macromolecule An injection which results in the evolution of heat exothermic within the sample cell causes a negative change in the DP power since the heat evolved chemically provides heat that the DP feedback 1s no longer required to provide The opposite is true for endothermic reactions Since the DP has units of power the time integral of the peak yields a measurement of thermal energy dH This heat is released or absorbed in direct proportion to the amount of binding that occurs When the macromolecule in the cell becomes saturated with added ligand the heat signal diminishes until only the background heat of dilution is observed With the 1T C2p system the entire experiment takes place under computer control The user inputs the experimental parameters temperature number of injections injection volume
42. rimental Curve a Outside of Optimal Options E Use Ka 17M 4 Time 3 Plat amp NDH The status bar just below the menus reads Thermostatting ITC 30 C which is the current status of the instrument When the system first loads this bar will read System Initiation Please Wait After a few seconds the system will begin heating or cooling to the preset temperature Once the instrument reaches the set temperature it will thermostat at that temperature To the right of the status bar the Time Left box During an experiment this box will show the time left until the end of the run When the software is first started by default the Experimental Design tab is selected this contains the simple run controls These let the user make a guess at N Kd and Delta H and have the software automatically design the experiment The Advanced Experimental Design tab contains more direct controls for the more advanced user This tab should be familiar to users of the VP line of instruments The Instrument Controls tab allows the user to name the output files choose post run analysis options and start and stop the run The Real Time Plot tab shows the data currently being generated The Setup tab contains various options and preferences 2 2 Experimental Design amp ITC200 Default User System ITC Options Help T hermostatting ITC 30 C Time Left fF r V r i gt Load Run File Save Run File Experiment
43. s and the computer carries out the experiment Origin software is then used to analyze the ITC data using fitting models to calculate reaction stoichiometry n binding constant Ka enthalpy AH and entropy AS 1 2 Parts and Accessories The main parts of the system are the cell unit and controller The tower on top of the 1TCy00 cell unit guides the motion of the pipette which holds the titration syringe The pipette has three positions cleaning loading and run positions It may also be placed in a neutral or rest position inbetween the other positions A glass 0 5 mL Hamilton loading syringe is used to fill and rinse the sample and reference cells The cell cleaning apparatus consisting of a small plastic barrel and soft tubing is used to run liquid through the sample cell for rinsing and basic cleaning A screw and ferrule fitting attaches to the pipette for rinsing and filling Also a thin piece of wire 1s provided for vigorous cleaning of the titration syringe tip Several other tools are provided for removal and replacement of the titration syringe and pipette tip Complete System Controller Cell Tower and Pipette Titration Syringe Soft Grip Tweezers Hamilton Loading Syring Cleaning Apparatus Pipette Tip Cleaning Wire Small Tipped Tweezers AW AN Nw AN yr i NA AN ann AN My Wh win 13 VENN 1 3 Software In order for the system to initialize properly all components must be powered up in the c
44. s attention to the risk of injury or contains safety notes and warnings Symbols used on the 1TC gt 00 instrument gt Caution Read the instruction manual before operating Instrument power supply ON Instrument power supply OFF Protective Ground earth Terminal Operational Safety The points below are intended to enhance your safety awareness and to draw your attention to risks which only you the operator can prevent While MicroCal works to ensure that the instrument is designed and tested to be as safe as possible proper handling is also critical The operators should be responsible people trained in basic laboratory protocol and they should be familiar with the possible hazards before operating this instrument All instrument modifications should be performed only by personnel trained by MicroCal Equipment damage personal injury or even death may result if this equipment is operated altered or maintained by untrained personnel or in an irresponsible or improper manner Provide proper electrical power to the instrument This should be 110 240 Volt 50 60 Hertz alternating current with a Ground Fault Circuit Interrupter GFCI Most power strips including those provided by MicroCal contain a GFCI All power plugs and cords should be 3 prong grounded cables or outlets Replace fuses ONLY with 3 15 Amp 250 Volt Time Delay Fuses Several spare fuses are provided with the original shipment 1 Repairs alter
45. s started the first operation that the 1TC gt 200 will perform is to heat or cool the cells and jacket to the temperature required for the experimental run This is accomplished when the cell temperature is within 0 1 degrees of the entered temperature and the DeltaT temperature difference between cells and adiabatic jacket is less than 0 001 C To save equilibration time it is usually best to set the 1TC20 thermostat temperature to the same as the starting temperature of the ensuing run or slightly colder When filling the cells with solution prior to arun the sample being loaded should always be colder than the desired experimental temperature in order to minimize the amount of time required to equilibrate the ITC Loading solution that is hotter than the desired run temperature will cause for lengthy equilibrations and or a run temperature that is slightly higher than requested Once the cells and jacket have reached the starting temperature the ITC will enter the second prerun state the prerun thermostat state The purpose of this state 1s to maintain the starting temperature for the requested period of time This allows time for thermal gradients within the ITC cell thermal core to minimize before the experiment starts and will reduce their effect during the experiment The default duration of the prerun thermostat state is 5 minutes After thermostatting the ITC instrument will enter the final baseline equilibration or the stirring stat
46. the same range of the display but always displaying the most recently plotted data Click OK to exit and save any changes or Cancel to exit without saving any changes DP Scale Controls FA Auto View 1 Auto View 2 Saved View 1 Saved View 2 Edit ranges Axis Rescale Ranges Options OK Cancel Full Scale Auto View 1 0 1 Full Scale Auto View 2 fi Saved View 1 Y Miji 000 Saved View 1 Y Max fi Saved View2YMin 10 Saved View 2 Y Max fo o X Axis Options Rescale Disabled The ITC Main Display shows the same data channels as the 1TC20 software cell temperature DP and DT The status bar shows the current state of the instrument and 1s the same as the 1T C gt 99 software status bar In off 2 9 Origin Data Analysis Tutorial 1 Open a copy of Origin for ITC data analysis Click on the Read Data button at the top left of the window 2 In the new window select your CaCl EDTA file and click Open Origin will load the data into a worksheet and calculate and display the integrated heat for each peak delta H 3 Ifyou did not enter the concentrations earlier click on the Concentration at the top left of the window In the popup window enter the concentrations and click OK 4 Click on the Remove Bad Data button just below the concentration button Click on the first data point and press enter to remove the point This first point is always smaller than the su
47. tion Check 49 General How To Use This Manual This manual describes the 1T C200 operation software and maintenance Section I contains general descriptions of the system its parts and tools the software how to perform a run and the tutorials These tutorials are recommended to every new user to acquaint them with the software and methods and to ensure optimal performance from the instrument Section 2 describes the software in greater detail Section 3 describes routine maintenance that users may need to perform from basic cleaning to periodic calibration checks to removal and replacement of parts How to Get Help from MicroCal The service department at MicroCal LLC is available to help you during normal business hours Monday through Friday from 8 00 AM to 5 00 PM EST The European office is available during normal business hours Monday through Friday from 9 00 AM to 5 00 PM GMT Service personnel may be contacted by e mail phone or fax with preference being phone or e mail When e mailing MicroCal for technical assistance if possible please attach a recent data file s 1tc raw ITC data file that demonstrates the problem Also please include all details that may be relevant to the problem For instances where the problem or question relates to post run data analysis it is best to attach both the raw data file 1tc and the Origin project file opj generated during data analysis There are two
48. tiply the cell concentration by n and by 10 15 for the syringe concentration Again for very tight binders the concentration should be on the low side and for weak binders this value should be on the high side The minimum and maximum concentrations that are commonly used in the 1TC2 are 3uM to 500uM These are rules of thumb and not absolute However if Ka and n are unknown a best guess should provide enough data to fine tune the parameters An analysis of the data from the run will show the average peak size approximate n and a better estimate of Ka Either the concentrations or the injection volumes should be adjusted so that the large initial peaks are 1 4 uCal second in height The peak height is roughly linearly proportional to each concentration and the injection size The refined estimate of Ka allows for an adjustment of the cell and syringe concentrations As an example assume we have an enzyme in the cell with a Ka of 0 000001 The concentration of enzyme should therefore be 10 Ka 0 00001 M or 10 uM Since this is a reasonably average Ka no further adjustment 1s required Assuming a stoichiometry of 1 the concentration of inhibitor in the 40 ul syringe should be 10 15 fold higher than the concentration of the enzyme in the cell or 10 uM 10 100 uM to 10 uM 15 150 uM 2 4 Instrument Controls dd ITC200 Default User MX T hermostatting ITC 30 C Time Left fF System ITC Options Help gt Load Run Fil
49. uid in the tubing and disconnect the tubing from the fill port Click the Purge Refill button 2 3 times to move the pipette tip down and back up again dislodging any air bubbles on the glass sides of the syringe Loading the Cell To load the sample cell gently insert the glass Hamilton syringe into the left sample cell until it touches the bottom Pull up on the plunger until bubbles are being pulled from the cell and there is no more liquid Remove and empty the syringe Clean the syringe if necessary Pull approximately 300 uL of sample into the syringe and tap the syringe glass so that all air is at the top volume of the syringe Do not allow air to be put into the cells After removing the bubbles insert the syringe into the cell and gently touch the bottom of the cell with the tip of the syringe needle Raise the needle tip about 1 mm off the bottom of the cell and hold it there until finished filling Do not raise the syringe during the filling process Slowly inject solution into the cell until it spills out the top of the cell stem Finish the filling with several small abrupt spurts of solution to dislodge any bubbles in the cells Finally lift the tip of the syringe to the cell port just below the visible portion of the cell port and find the ledge that 1s formed where the cell stem meets the cell port Place the syringe on the ledge at the top of the metal cell stem and remove the excess solution If the reference cell needs refilli
50. ulated Wwsla Mean Energy Injection 0 34863uCal SD 0 08232 uCal Results Within Specifications Line 2 of the script is the instrument serial number line 3 is the date and time that the report was generated line 5 is the injection size and lines 7 and 8 are instrument specifications Origin manipulates the offsets so that multiple water runs can be displayed without overlapping Line 10 gives the data file name and the mean and standard deviation of the energy per injection The last line will say Results Within Specifications or Results Outside of Specifications If the results are not within specifications you may want to run the tutorial again CaCL EDTA Tutorial This tutorial uses the standard sample kits provided with the instrument when it is shipped Kits can also be obtained from MicroCal at any time 1 Load the titration syringe with CaCL The sample cell must be rinsed with EDTA in order to obtain an accurate fitted value for stoichiometry Fill the sample cell with EDTA and allow the solution to remain in the cell After 5 minutes remove the EDTA solution from the cell Load a fresh volume of EDTA in to the sample cell 2 Set the run parameters A set of parameters 1s provided with the sample 3 Name the file and click Start to begin the experiment 4 Once all injections have been completed clean and dry the syringe Use the cleaning apparatus to clean the sample cell See section 2 9 Origin Data Analysis T
51. user convenience in viewing data generated by the ITC200 Following is a description of each of these sets of tools Buttons for Data Display This group of buttons allows you to rescale the y axis by simply clicking on a single button Rescale To Show All shows all the data currently on the plot Auto View 1 will rescale the y axis so that the last DP data point plotted will be centered on a full scale determined by the entry in the Full Scale Auto View 1 text box in the Edit Ranges box see description below By default it is 0 1uCal sec Auto View 2 rescales the y axis to a second scale centered on the last DP data point plotted By default it is 1uCal sec Saved View 1 rescales the y axis to a specific range specified in the Edit Range box By default the range is 1 to luCal sec Saved View 2 rescales the y axis to a second specific range By default the range is 10 to 10uCal sec Edit Ranges opens a window that contains parameters to be used with the aforementioned buttons addition to the Auto View and Saved View entries which are described above the X Axis Options drop down list box controls the automatic re scaling of the x axis when the data exceeds the X Axis time display range Disabled Takes no action Current data may be plotted screen Rescale Extends the maximum X Axis setting by 25 resulting in a larger display range Scroll Extends the minimum and maximum X Axis setting by 25 preserving
52. utorial for analysis of the results NOTE Detailed instructions concerning sample loading and data collection using the standard CaCl EDTA kit are provided with each kit Section 2 iT C2 Software and Origin This section details the features of the 1TC2oo software This software provides the user an interface to control the 11C gt 00 instrument and perform an experiment An optional copy of Origin can be used for plotting the data received from the 1TC x instrument A separate copy of Origin Origin for ITC is used for analyzing data 2 1 iT Cv Software Overview The 1TC209 software contains drop down menus a status bar and several tabs The drop down menus control global system functions The status bar displays the current status of the instrument Each tab contains controls for different areas of instrument control dt ITC200 Default User System ITC Options Help T hermostatting ITC 30 C Time Left gt q 9 Lay ml Load Run File Save Run File Experimental Design Advanced Experimental Design Real Time Plot fs Setup 1 Experimental Mode Results Please chooze hi Cell uh Change 0 Help Syringe utd Change o Hep E Change E Help Expected N Moles Mole Estimated Kd M Help Delta H kcale Mole C Yalue Legend Optimal 5 500 Temperature 30 Degrees C Data File Name Outside of Optimal Range default tc 15 500 1000 Update Expe
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