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1. DP DeltaT 1 JTemp ipu PEZ 10000 10 10 ITC Cell Status DP pCal sec 4 096 DT C 0 003 8 8 5000 T ITC Numeric Display 6 6 Display Mode Z 0 Rescale To Show All a 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 Y s Time sec j Na Edit ranges m IRR m le A eL HENAO x y VPITCPLOT OPJ Name Type J Spata Workshee lotr Zr anb v i gt 1 Datal_DP 1 0 Plot Radian The ITC cell status the ITC gt 200 numeric display and the buttons for ITC299 data tools as indicated above have been added for user convenience in viewing data generated by the ITC oo Following is a description of each of these sets of tools 38 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 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 lu Cal 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
2. U The operator should always follow proper laboratory procedures in handling and disposing of volatile or hazardous solutions 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 U 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 T C gt 00 instrument should always be moved in its normal operating orientation Other orientations will subject delicate sensors inside the instrument to stress The 1TC 5oo cells are constructed out of Hastalloy Strong acids should be avoided Please refer to the accompanying material analysis booklet for further information This instrument 1s 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 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 500 1500 RPM Physical Specifications Cell Material Hastelloy Alloy C 276 Dimensions Calorimeter 6 x 13 x 10 2 Controller 15 x 16 x71 Monitor 17 x 16x 555 Washing Module 7 4 x 6
3. MicroCal 22 Industrial Drive East Northampton MA 01060 USA Tel 413 586 7720 800 633 3115 Fax 413 586 0149 Support microcalorimetry com Binding Stability Kinetics www microcal com ITC599 Microcalorimeter User s Manual MAU501460 Rev D Ultrasensitive Calorimetry for the Life Science NE MicroCal Table Of Contents Generals tnim ntium nA 1 HowTo Use This Mantal meen Pe tata A RA PTO EC ues l Howto Get SO RST Ee LET E E 2 MicroGal ELC Contact into v a 2 SYDOST E it bellies hela della ab totu tad sae 4 Operational Salet y scs pn tes vtri te dices ls ep iu a 5 Instrument pecan TT T P rtotimalceSDectPcalbOLis usd toda AAA A A AAA oe 7 Physical OME CHIC AL T il Electrical PC CHI CATIONS AA AA IT 7 Aimospheric SPECICATION aariaa bees nebat Gera ees Sele m E A E N 7 Section 1 Introduction to the 11 C00 ccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccees 9 ul The Basics or TG aresoarra a iia tds 10 EZ Parts amd ACCESSOMeS cana Oto D ei ned Oa ences eae 12 ka DON T CRT EP T UU T Em 14 Opt ornal OPI pad o A A L Oo noes e reet peut 15 LA Bastcsor Perrormit g 3 Bliss a adco A i eee A fai aes 16 Load did 16 A Ib soci etos Bee kot rene Oo eR Orn Ee eRe rr nr ona Cnn Pp eT rr er poner E net re eee 16 ko R Se T Epen men S oec oR hte dott eda olde di adt etus 18 Water Water Tutorial meae asa ettet AAA EET d E eA CES 18 ACD TACO NERO m m 19 section 2 iT Czo Softwa
4. 3510 72797 Total 4 rounds in this sessi on 2 Levenber g Mar quar dt Successfully progressed 1 rounds Reduced Chi sqr 3510 69257 Total 5 rounds in thi s sessi on 3 Levenber g Mar quar dt Successfully progressed 1 rounds Reduced Chi sar 3510 69257 Total 6 rounds in this sessi on 4 Levenber g Mar quar dt Chi sqr is not reduced Reduced Chi sar 3510 69257 Total 6 rounds in this sessi on Click Done A textbox will appear on the graph window 7 Select Origin menu ITC 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 40 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 41 3 1 Cleaning the Cells and Syringe Cell Cleaning The 1TC oo 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 i
5. Help Total Heat y Cal n Help Cell pM aa Help Syringe pl Gangs lla Heb C Value Change r Help Estimated Values N Help Kd M Help AH kcalz Mole Help delta H C alimale Legend Si Within Optimal Range Temperature 55 Degrees E Data File Name O default itc Update Experimental Curve i Ei Outside Suggested Range Options Outside Optimal Range C Use Ka 17M ee AH 0 4 0 6 Molar Ratio When the instrument first boots up the line just below the menus reads System Initialization 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 If the instrument is not attached or not turned on the program will open into Demo Mode in which the user can see and manipulate the program but it will not attempt to control the 1TC290 To the right of the status bar the Time Left box during a run will show the time left until the end of the run 14 When the software is first started the Experimental Design tab is selected this contains the simple run controls Experimental Mode can be Highest Quality Minimum Protein or High Speed The expected n Kg 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
6. In the Feedback Mode Gain section the three available modes are none low or high High 1s 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 1T C oo 1 e kinetics metabolic rates might benefit from using the passive or low gain feedback modes 26 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 Parameters 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 is 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 injection
7. 1s held by the arm and connected to the tower by a black cable First disconnect the black plastic connector by removing two screws see below and pulling the connector loose This requires a 0 050 hex driver Loosen the screw on the side of the arm to remove the pipette E Yellow circles indicate screws securing connetor To reinsert the pipette place the pipette in the socket on the arm and tighten the screw Insert the cable into its slot and tighten the two screws It is a good idea to recalibrate the open fill port position once the pipette has been replaced see section 3 5 46 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 knife provided with the 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 1mage 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 1s 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 47 3 5 Pipette Calibration The pipette may need to be recalibra
8. 2 Usinga clean 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 syringe with water 3 Within the 1TC oo software activate the extended data mode by clicking the Extended Data Mode box in the Real Time Plot tab While the software 1s in the extended data mode the ITC data files will contain all available information produced by the 1TC200 This additional information will often help the MicroCal technician diagnose problems 4 Carry out a minimum of 20 2uL 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 iicrocal 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 Micr
9. Col ater Bitsed Lona 64 Detergent soak and Rinse UO ata sc e IR TERR EE ete IRE e uet dci 65 yace Washi SOLE esit oat estes a tonius 67 NT W ISI bog PRE PEO O matu teta eee ep terat 68 Dy MIN ct EP 68 Repiacine Vials and H oov er Rh RERO sigue TE 70 How To Use This Manual This manual describes the 1T C599 operation software and maintenance Section 1 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 Section 4 describes the Washing Module s functionality and controls 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 1s 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 A
10. Delat Read 8333333 Min Safe Temp 3 T Shield T Read 2849034 0 752929 Max Safe Temp zT im ATP Read B O DP FB Made A D Slope D A Slope DP Passive FB DEBE ITC Injector Coefficients DP Low Gr FB Sara Steps Fer Inch 256000 OF Mid Gn FB 29862 Max Safe Postition 260972 steps DP High Gn FB i Syringe Constant 431 ul in Siapa Offset Cao Na EO aN 0033303 0 027142 APM 1 RPM 2 RPM 3 RPM 4 ATP Output 16 6 ATP Time Shift EU 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 is disabled the system will go through an additional prerun state a non stirring equilibration period before stirring is 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 36 E ITC200 Default User System ITC Ns Help Dryin System Coefficients le u
11. Start in Advanced Made Lay ITC Equilibration Options K Load Run File Taa RT ley Advanced User Mode Advanced Experimental Design The Accessory Station menu option allows the user to switch to Advanced User Mode in which the Accessory Station routines do not prompt the user to move components between cleaning stages E ITC200 Default User ITC Options System Contact Info About Experimental Du I lane C nts rm The Help gt Contact Info menu option provides contact information for MicroCal support The About menu option provides specific details about this version of software 37 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 ITC oo data display s Origin 7 C Origin7OWPITCPLOT OPJ Read Only Plot File Edit View Graph Data Analysis Tools Format Window Help _ ax x e a ESSE el ala e als Al Aloe ASE e Es lele lol MAA E T UN 1 LLqqZ U Te Current Status Thermostatting ITC 30 C Main Display JTemp1
12. bottom of the cell and hold it there until finished filling Do not raise the syringe during the filling process Slowly inject solution into 16 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 is 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 refilling follow the same procedure as for the sample cell Ledge Formed Where Cell Stem Meets Plastic Place Where Overflow Reservoir Bubbles Are Trapped 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 1T C00 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 on the bar of buttons across the top 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 equi
13. 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 1s 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 K4 or K and to choose whether to view the simulation plot using raw heat per injection DH or the heat normalized to the molar ratio NDH 24 2 3 Advanced Experimental Design The advanced experimental design tab may be used in addition to the Experimental Design tab for operating the iTC20 instrument Below is a description and suggested values for each of the run parameters displayed in this tab PASE E Eee ORE Default User System ITC Options Help Thermostatting ITC 25 T Time Left Load Run File Save Run File Experimental Parameters mc njection Parameters TER nea MEME he nant cane vonesantranenentsguenakesanenay 2 24 Cell Temperature C ON 4 48 Reference Power uCal sec 0 1225 reer etre o eee ee M L TTE TTTUTIT desa IAN o Syringe Concentration mM o cr msc 2 Edit Mode Cell Concentration mM 4 All Same lt gt Unique l
14. 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 containing 100uL of your sample 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 Connect the threaded end of the tubing from the Washing Module to the pipette fill port 1f necessary it should still be in place after cleaning and drying of the syringe In the Instrument Controls tab click Syringe Fill The software will prompt the user to move the pipette as necessary and the Washing Module will fill the syringe See section 4 3 for more information 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 1s 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
15. 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 In 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 Saved View 1 Y Min E Disabled Takes no action Current data may be plotted Saved View 1 Y Max i off screen Saved View 2 Y Min j10 Rescale Extends the maximum X Axis setting by 25 Saved View 2 Y Max HT resulting in a larger display range Scroll Extends the minimum and maximum X Axis setting by 25 preserving the same range of the display but always displaying the most recently plotted 1 DP Scale Controls T Axis Rescale Ranges Options OK Full Scale Auto View 1 0 1 Full Scale Auto View 2 fi X Axis Options Rescale Auto View 1 Auto View 2 Saved View 1 Saved View 2 Edit ranges L Cancel data Click OK to exit and save any changes or Cancel to exit without saving any changes The ITC Main Display shows the same data channels as the 1T C5oo software cell temperature DP and DT The status bar shows the current state of the instrument and 1s the same as the 1T C oo software status bar 39 2 9 Origin Data Analysis Tutorial 1 Open a copy of Origin for ITC
16. x 54 Weight pounds Calorimeter 16 Controller 20 Monitor 9 5 Washing Module 6 For operation the 1T C5oo with a computer controller requires about 1 meter of normal bench space ca 70 cm wide The ThermoVac degassing station will require another 25 cm of bench space 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 4 00A 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 cells Humidity 10 90 Atmospheric Pressure 500 1060 HPa Section 1 Introduction to the iT C 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 it includes several suggested tutorial experiments 1 1 The Basics of ITC The 1TC oo 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 r
17. 498 0 3 DP 50 5 100 150 300 Time sec 1 DP Tools oom 0 05 Zoom t 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 1s 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 32 2 6 Setup PASE E er ORE Default User System ITC Options Help Thermostatting ITC 25 T Time Left fF E E z gt l lay ETT ess Load Run File Save Run File User Setup Data File Path C ITC200 data Setup File Path C ITC200 setup Init Setup File astunlin 3 OOtOt lt C S st S Cells Boot Temp 25 C Save User Ge dd User Curr
18. I All power plugs and cords should be 3 prong grounded cables or outlets The Washing Module may only be powered by the power supply provided with the Unit t s Intermittent operation of the Washing Module is required so that the valves of Washing Module are on for a maximum of 20 minutes then off for 30 minutes Software limits the valve on period to 10 minutes before an experiment so this does not affect normal operation of the instrument Replace fuses ONLY with 4 00 Amp 250 Volt Time Delay Fuses Several spare fuses are provided with the original shipment U e Repairs alterations 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 U e 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 1s 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
19. P default tk 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 51 3 7 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 1TC5oo software This procedure requires a Control Company digital meter and submersible probe or equivalent 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 1T C599 software to set the
20. 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 1T C oo 1s 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 1T C5oo 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 is started the first operation that the 1TCooo will perform is to heat or cool the cells and Jacket to the temperature r
21. a non administrator to use this software read write privileges must be set for all users for the ITC200 and Origin70 folders including all subfolders The folder properties must allow Read amp Execute List Folder Contents Read and Write as shown below ITC200 Properties Origin70 Properties General Sharing Security Customize General Sharing Security Customize Group or user names Group ar user names a DC1 hdi al E Administrator USER 7E FCIDWNJ2 Administrator E Administrators DC1589063 Administrators Administrators USER 7E 7CJDWNJ2 Administrators G CREATOR OWNER G CREATOR OWNER SYSTEM A Users DC15890634U sers CE SYSTEM Lasers LIL aeea leB Ser er Users USER 7E CID WJ AU sers Permissions for Users Aon a Permissions for Users Allow Dery Full Control Modify Read amp Execute List Folder Contents l Fead List Folder Contents Full Control Modify Read amp Execute write Read C al l m ERI nl K M rite For special permissions or for advanced settings Advanced l One e eren a Advanced For special permissions or for advanced settings click Advanced Is s S ISTE O Note Refer to the following Microsoft article to enable the security tab if it 15 not available http support microsoft com default aspx scid kb en us 290403 53 Section 4 Washing Module This Washing Module is provided with the 1T C5oo to help clean the sampl
22. als carefully pull down and out on the handle of the vial The vial should slide out To replace the vial insert the fill tubing and filter 1f applicable into the vial and slide it up and back into its slot The solvent vials should be filled with from left to right distilled water methanol and buffer If buffer is not desired for some reason this vial may also be filled with water The methanol should be removed and capped when the instrument is not in use as methanol is very volatile The vacuum waste tube must be unscrewed from its socket then pulled down and out To replace it slide the tube up and back and screw it back into its socket To replace a filter element first remove the solvent vial Hold the nut at the top of the filter and pull gently on the filter element to pull it off the tubing Push a new filter element into place and replace the solvent vial To replace the filter and nuts first remove the solvent vial Unscrew the filter nut from the nut holding it Pull the filter off of the tubing If the second nut also needs to be replaced pull it off as well 70 If the nut and ferrule were removed first insert the ferrule into the nut see picture The flat end of the ferrule should be out Slide the nut and ferrule onto the tubing and push them up about two inches 5cm Slide the filter and nut onto the tubing and tighten the screw to secure it Reinsert the solvent vial 71
23. anol is very volatile To install the vacuum tube slide the tube up and back and screw it into its socket 56 Attach the tubing from C5 to the wash bottle as shown below 57 4 2 Cell and Syringe Wash TC200 Default User System ITC Options Help gt Load Run File Save Run File play Run Param Update Run Parar Experimental Design Advanced Experimental Design ITE _ _ I e 4 Accessory Station Cells Passively Cooling to 25 C Thermostat Control a Set Point C m Cell and p Wash Open Fill Port lee Fill Close Fill Port Cell Buifer Rinse Set Jacket Temp Short A li Cell wafer Rinse Purge gt ReFill Long g l 25 12 Pulse Control Set Up A Pulse Size Distance Im Up Detergent S oak and Rinse Long uL S Syringe T Short Maintenance Syringe A Long 1 Remove Old Tip Dry 5 2 Install New Tip ta siiis 3 Mew Tip Installed Auto Postrun Data Analysis Properties Hun Type Experiment Control Estel Eie Mame Duration 300 Pulse On Control Subtract Options Control Run File Name Insert the cell cleaning apparatus into the sample cell see pictures below Be sure to push down until the device is firmly seated Attach the syringe connector to the syringe Click the Cell and Syringe Wash button in the Instrument Controls tab of the 1TC5oo software The software will prompt the user to be sure th
24. ard 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 CaClo The sample cell must be rinsed with EDTA 1n 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 is 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 Tutorial 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 19 Section 2 iT C Software and Origin This section details the features of the 1T C5oo software This software provides the user an interface to control the 1T C5oo instrument and perform an experiment An optional copy of Origin can be used for plotting the data received from the 1TC200 instrument A separate copy of Origin Origin for ITC 1s used for analyzing data 20 2 1 iT Cp Software Overview The 1TC oo software contains drop down menus a status bar and several tabs The drop down menus control global system functi
25. be clicked for the run parameter changes to take effect This button is available only when the 1TC oo 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 1gnored 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 although 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 1s 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 sett
26. 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 succeeding 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 Levenber g Mar quar dt Successfully progressed 4 rounds Reduced Chi sqr
27. 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 1T C oo software if desired 1s 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 1s 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 1s 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 range All four of these views can be edited through the Edit ranges button 15 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
28. e cell and titration syringe The software that controls it is located in the Instrument Controls tab Clicking one of the buttons will start a cleaning or rinsing routine with prompts to direct the user to move parts as necessary These prompts can be turned off under the Options menu and Accessory Station submenu by unselecting Advanced User Mode 54 4 1 Setup Place the Washing Module on top of the 1TC200 Plug in the power adapter Remove both USB cables from the back of the 1TC200 and plug one into the input port of the Washing Module Plug in both USB short cables The left port on the Washing Module is attached to the upper port on the 1TC200 and the right port is attached to the lower port Attach the tubing as shown below All the connectors and the cleaning device should already be attached to the tubing so that the tubing only needs to be connected 55 C1 to syringe fill port C2 to needle wash port Cato top of cleaning device 5 to waste CA to side of cleaning device Insert the vacuum tube and three solvent vials To install a vial insert the fill tubing and filter if applicable into the vial and slide it up and back into its slot The solvent vials should be filled with from left to right distilled water methanol and buffer If buffer is not desired for some reason this vial may also be filled with water The methanol should be removed and capped when the instrument is not in use as meth
29. e cleaning tubing is inserted The program will first execute the Cell Buffer Rinse Short procedure 58 Accessory Station Please place the wash tool in the cell Cancel Accessory Station Please move the pipette to the wash position and connect the syringe tubing Cancel 59 A prompt will appear after the cell wash portion of the sequence 1s complete The user may then load the cell Click OK to continue and clean the syringe The program then executes the Syringe Wash Short procedure The syringe cleaning takes about nine minutes Once that has completed the syringe may be loaded with sample Accessory Station You may remove the wash tool and fill the cells if desired Cancel 60 4 3 Syringe Fill Leave the tubing connected to the syringe Leave the syringe in the wash position or the rest position Click Syringe Load Accessory Station The plunger will move to the bottom of the syringe to prepare for loading This must be performed with the syringe NOT in the titrant to prevent turbulence and gassing of the sample The software will prompt the user to move the pipette to the titrant Accessory Station Please place the pipette in the titrant Cancel Click OK to continue The pipette tip will move filling the syringe After the tip reaches the open port position the pump will remove the air bubble reducing titrant usage The port will move to the close position and the
30. eactants Spin rates are user selectable the usual range is 500 to 1000 RPM The normal temperature operating range 1s 2 C to 80 C Wetted cell surfaces are Hastalloy which are resistant to most solutions however strong acids should be avoided sample and reference cells are accessible for filling and cleaning through the top of the unit The sample cell 1s 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 1s 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 Bangor zl Lead Sog Sensor LI EE I br dI il Injector E ill hij Ex tl MT Plunger lr 197 E 1 1 String amo syringe Kutar Shield Inner Shield Sample Call O I A Haler nca Gall Temperature 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 10 equilibrium Calibration of this signal 1s obtained electrically by administering a k
31. ect 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 Wat er Water Report VPI TC11 06 834 12 5 2006 08 42 23 Raw DP Data lOuL i nj ecti ons M t er W ter Speci fi cati ons Mean Energy I nj ection less than 1 5 uCal SD less than 0 15 uCal Baseline Offsets Have Been Mani pul at ed VWsla Mean Ener gy nj ecti on 0 34863uCal SD 0 08232 uCal Results Wthi n Speci fi cati ons Line 2 of the script 1s the instrument serial number line 3 1s the date and time that the report was generated line 5 1s 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 18 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 stand
32. en 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 is heat added to the sample cell by an exothermic reaction will result in a downward deflection in the baseline DP signal This downward deflection 1s 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 1s 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 1s used to determine the characteristic curve 3l 2 5 Real Time Plot amp 1TC200 Default User System ITC Options Help Thermostatting ITC 25 T Time Left fF Load Run File Save Run File Temp C DP UC als 25
33. ent User Default User Erase User Y Axis Scale Extended Data Mode TESI lt gt mCal min Export Current Data pCal sec lt gt watt 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 1TC299 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 1TC5oo 1s 1n 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 1TC299 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 33 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 specifie
34. equired 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 1T C5oo thermostat temperature to the same as the starting temperature of the ensuing run or slightly colder When filling the cells with solution prior to a run 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 1s hotter than the desired run temperature will cause for lengthy equilibrations and or a run temperature that 1s slightly higher than requested 30 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 1s 5 minutes After thermostatting the ITC instrument will enter the final baseline equilibration or the stirring state The syringe will start to rotate The start of the stirring will cause the DP signal will deflect downward and th
35. g 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 1s equilibrated and the resulting deflection used as a crude calibration assessment If Origin for real time plotting 1s enabled Origin will calculate the error If this is greater than 1 please see Section 3 6 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 Accessory Station controls run the automated cleaning routines for the Washing Module The Stop button at the bottom of the panel will abort whatever routine 1s in process See Section 4 for more information 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 postrun data analysis is selected under the Options menu Run Type tells the computer whether this run 1s an experimental run or
36. ied Individual pulse parameters are entered by first selecting a pulse or multiple pulses then entering 50 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 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 is reported as higher than 1 please contact MicroCal For a more rigorous analysis once the calibration 1s done and the system is thermostatting again open the ITC Calibrations project Click on the Y Axis Calibration DP D
37. ing 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 u Cal 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 1s 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 if desired see section 2 2 or refer to Calculating Concentrations later in this section for more information If the Experimental 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 19 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
38. is Properties Control Subtract Options Heal Time Plot Accessory Statin Cell and T pringe Wash 5 Ed Fill Cell bale er ies Cell Rinze ong s Detergent Soak and Rinse Long Syringe m Short Syringe es Long Dry hoe oom Control Run File Name The Instrument Controls tab contains the controls for direct operation of the instrument At the top of the window the user can Start the run using whatever parameters are currently present in the Experimental Design or Advanced Experimental Design tabs Before clicking this button it 1s 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 1TC299 thermostating idle state Pre thermostatting the 1TC200 and samples at the run temperature will result in 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 29 The Pulse Control section allows for manually administerin
39. item allows the user to switch to Single Injection Mode SIM This corresponds to the High Speed mode in the Experimental Design Tab The Pipette Tools menu option opens a popup window for adjusting syringe calibrations See section 3 5 for more information The Start ITC Calibration Run menu item is 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 35 4 ITC200 Default User System ITC sleet Help Dryin System Coefficients imd sy Start in Advanced Mode lay ITC Equilibr ation Options Z w Fast Eoul Load Run File Accessory Station kw Auto Mode Advanced Expermental Design System Coefficients opens a popup window with the most critical of the software calibration constants This 1s 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 amp System Coefficients YPITC12 OF 030 Cell Parameters _ 1 0 Channel Coefficients Cell Volume 92 Slope Offset Ref Cal Htr 158000 Jacket T Read 2351131 0 83274 Samp Cal Htr 769000
40. librate to that temperature start the titration syringe stirring wait until the DP signal 1s 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 17 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 l 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 1s located 1n the Origin directory at C Origin70 ITCCalibrations OPJ Click on the Water Water Report button A window will pop up asking which files to analyze sel
41. ll prompt the user to make sure that the tubing 1s connected Accessory Station Please move the pipette to the wash position and connect the syringe tubing Cancel Click OK The Washing Module will perform a short washing routine approximately 8 minutes It will rinse the syringe first with water and then with methanol It will then pull vacuum through the syringe for several minutes to dry it 67 4 8 Syringe Wash Long Connect the syringe tubing to the syringe fill port Click Syringe Wash Long The software will prompt the user to make sure that the tubing 1s connected Accessory Station Please move the pipette to the wash position and connect the syringe tubing Click OK The Washing Module will perform a more thorough washing routine approximately 8 5 minutes It will rinse the syringe first with water and then with methanol It will then pull vacuum through the syringe for several minutes to dry it 4 9 Dry Syringe 68 This function is used when the syringe has been cleaned manually and left with methanol in it Attach the syringe connector to the syringe and place the pipette in the wash position Click the Dry Syringe button Accessory Station Please connect the syringe tubing and move the pipette to the wash position Cancel The washing module will pull vacuum through the syringe for several minutes to dry it 69 4 10 Replacing Vials and Filters To remove one of the solvent vi
42. lso please include all details that may be relevant to the problem For instances where the problem or question relates to post run data analysis 1t is best to attach both the raw data file 1tc and the Origin project file opj generated during data analysis There are two general categories of troubleshooting for the 1TC gt 00 and its operation The most extreme category is when a system is not working at all Problems that prevent users from operating the 1T C5oo require immediate consultation with a MicroCal technician Customers should not attempt to repair the 1T C599 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 C5oo instrument is functioning but 1s 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 1s 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 Washing Module to perform a Cell Water Rinse Long Discretion may call for a more rigorous cleaning procedure
43. nds 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 1T C5oo software is able to collect and save Calculating Concentrations The easiest way to determine a good starting concentration for a system is to use the Experimental Design tab of the 1T C259 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 Kg 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 Multiply 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 1TC299 are 3uM to 500uM 24 These are rules of thumb and not absolute However 1f Kg and n are unknown a best guess should provide enough data to fine tune the parameters An analysis of the da
44. nown 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 is 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 11 C gt 00 system the entire experiment takes place under computer control The user inputs the experimental parameters temperature number of injections injection volumes and the computer carries out the experiment Origin software 1s then used to analyze the ITC data using fitting models to calculate reaction stoichiometry n binding constant Ka enthalpy AH and entropy AS 11 1 2 Parts and Accessories The main parts of the system are the cell unit and controller The tower on top of the 1T C5oo cell unit guides the motion of the pipette which holds the titration syringe The pipette has fou
45. ntly until it slides down into the storage case The softgrip tweezers may help with this Move the pipette out of the way and screw the cap onto the storage case Remove it from the loading hole 44 See images 5 10 for illustrated syringe replacement Insert the storage case into the loading hole and unscrew the top Position the pipette over it Slide the case up until the syringe glass enters the bottom of the pipette Let the case drop and 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 Be very careful as if the syringe 1s not all the way up the needle will catch on the edge of the storage case likely bending the needle and making it unusable Move the pipette to the cleaning hole and turn the inner metal syringe holder to the left several turns so that the bottom nut is engaged but not fully tightened Move the pipette to the loading hole and insert the fill port adapter Make sure that it screws in easily 1f it does not loosen the bottom nut until it does With the adapter in place tighten the nut This ensures that the syringe 1s lined up properly within the pipette Disconnect the adapter 45 3 3 Removing Pipette The pipette
46. oCal 1s a registered trademark of MicroCal LLC Northampton MA Windows is a registered trademark of Microsoft Corporation Symbols Symbols used in this manual i Warns the user of possible damage to the unit draws attention to the risk of injury or contains safety notes and warnings Symbols used on the 1TC200 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 P C gt 200 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 GFC
47. ommended 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 Zu er Default User stem BEES Options Help Print Save As Text Run Parameters i Drying Sim Made E Pipette Tools Start ITC Calibration Run Y Axis Check Dad Ri Experimental Design Advanced Experimental Design ITC Y Axis Calibration Sie File DP Calibration Parameters Experimental Temp 30 Data FileName defaultitk Number of Pulses g Reference Power 55 Filter Period 2 Stirring Speed 1000 Feedback Mode Gain None Low High Pulse 7 Euan AN Miisa ENT 3 lec CAN Pulses Calibration Power uL al sec 11 Pulse Duration sec 300 Pulse Spacing sec 600 Start Hun Cancel To begin the y axis calibration check procedure select 1TC299 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 modif
48. one 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 removed 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 42 glass will help Continue to insert the wire until it emerges from the tip of the needle see image below Carefully pull the wire back through the needle and glass bore 43 AUN CAS aw Y wos Y stp ES V y SN S RON ws qp 4 ANE NW WW Y e quet yn E AS o ue 3 2 Changing Injection Syringes Clean and dry the syringe before removal See images 1 4 below for illustrated removal Remove sample tube from loading hole and disconnect the cleaning apparatus Insert the syringe storage case into the loading hole 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 nut that holds the syringe in the pipette Once the nut is loose move the pipette to the loading hole Use the manual pipette controls to lower the pipette tip about 0 3 inches Without the bottom nut the syringe will move down with the pipette tip Pull down on the syringe ge
49. ons The status bar displays the current status of the instrument Each tab contains controls for different areas of instrument control PASE E Een ORE Default User System ITC Options Help gt Load Run File Save Run File Stark Experimental Design Advanced Experimental Design Heal Time Plot fs Setup 1 Experimental Mode Results Please choose lt Help Total Heat y Cal n Help Cell uM Change n Help Syringe pl Change o Heb C Value oc Help Estimated Values Mo Help Kd M Help AH kcalz Mole Help delta H Cal mole Legend Temperature 2n Degrees C Ci Within Optimal Range Data File Name Outside Optimal Range default itc Update Experimental Curve i E Outside Suggested Range Options Use Ka 1 M Oe AH 04 Molar Ratio The status bar just below the menus reads Heating To 25 C which is the current status of the instrument When the system first loads this bar will read System Initialization 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 thi
50. ontains the simple run controls Experimental Mode can be Highest Quality Minimum Protein or High Speed Highest Quality uses 20 injections These parameters should produce data that is clear and easier to fit Minimum Protein uses fewer injections only 10 The result of these parameters will be the use of the least amount of sample necessary for a successful titration The High Speed mode will do one single longer injection Single Injection Mode SIM The expected n Kg 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 Kg for their system clicking the Help button causes the software to prompt for the type of compound in cell and syringe It will then make a guess as to the Kg 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 29 have values for the cell and syringe concentrations The calculated C value 1s listed below its background is color coded The c value predicts the shape or sigmoidicity of the curve Optimal values for C are between 5 and 500 green values between 1 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
51. or 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 34 2 7 Menu Options The main drop down menus at the top of the 1TC5oo 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 1TC oo will also be shut down Power to the ITC will not be restored until the application is run again T ITC200 Default ITC 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 1T C gt 200 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 record keeping 11C200 Default User stem BASS Options Help Print Save As Text Run Parameters E Drying Sim Mode E Pipette Tools Stark ITC Calibration Run Y Axis Check Dad Ri Experimental Design Advanced Experimental Design The Sim Mode menu
52. r positions cleaning loading run and rest 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 1s 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 is 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 Pipette and Washing Module Titration Syringe Soft Grip Tweezers 12 Hamilton Loading Syringe Cleaning Apparatus Pipette Tip Cleaning Wire Small Tipped Tweezers 13 1 3 Software In order for the system to initialize properly all components must be powered up in the correct order First boot up the computer and log in to Windows Once Windows has started power the 1TC5oo by operating the switch at the rear of the unit After several seconds open the IT C5oo software If the option is selected a real time copy of Origin will open automatically as well as the 1T C oo control software e TC200 Default User System ITC Options Help gt Load Run File Save Run File Experimental Design Advanced Experimental Design Heal Time Plot sep O 1 Experimental Mode Results Please choose lt
53. re and Origin este eeu oen or eee eaae e eee ye aeo Fue eee Ei pua ote eee esee USE Ree ae vae 20 2 1 TEC 56 Software Overview eiii liada silla 21 2 2 Expertiienral CS Ti c ooo cob Condo MEE 23 2 9 Advanced Experimental Destaca 25 Calculating Concentrations pc boosh a ad a NIE dd 27 24 Insttument C oDtFOls eser du dicata as 29 Statt s OL A O a ne en ee 30 2 RealTime LOU etie tostuedu meuf o deua it tl fuit sco npe E ctas 32 M ce 1P AA Ada 33 Ded J NICO UST T mmm 35 2 0 Ono Real Time Display oen rte oda S euet leen P deles RR 38 2 9 Qnem Data Analysis Tutoria bemori n te rente daa 40 Section 3 Instrument Maintenance ccccccsccsssccscsssccssccsscccsccsscccsccsscccsccsscccsccsscccscessccesccssees 41 3 L Cleaning the Cells and SAO uie oer See n deducts ab augdsn duu asesengdndeeducuaaes 42 Cell C Ie atti ee octopus I IM IM MI IM IMEEM ILE I PM EU 42 O A O N 42 3 2 Chanpino Injection SV IIS ES idad a 44 355 REMOS e Ge o n roD DUM OMM EI IM nU E 46 34 Eur ulum E 47 3 9 IDPIpette CaltbEatoiiuca ceste rt occa oed uisaute uaa II Ua Man eae aed 48 3 0 Yeas dll Dra CA castor ncaa Uo euf e Ebedo diced geht 50 5 7 Temperature AH Dial OM C Dee Rs caedes ernst gotten tddi 52 3 8 Configuring an 1T C5oo Controller for N eworidng esse eee ee eee eee 53 SECHON 4 Washing dr 54 4 10 Vis MM T M 55 CALA a STT TI de 58 SA cm 61 Cell D trer Rias MOR ia 63
54. s 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 1TC5oo 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 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 seco
55. s time with water using greater quantities This 1s especially useful after a detergent soak to be sure of removing all of the detergent 64 4 6 Detergent Soak and Rinse Long Click the Detergent Soak and Rinse Long button and follow the prompts to load 5 Contrad70 Accessory Station Please load the sample cell with detergent such as Contrad 7ll The system temperature will be 50 degrees for 30 mins message will appear when itis safe to unload the cleanser A cell rinse will be performed next Cancel The cells will heat to 50 C for half an hour to soak Once they have cooled the software will prompt the user to remove the Contrad70 and insert the cleaning device for rinsing and then to connect the pipette connector to its holder Be sure that the cells have cooled before removing the Contrad70 as hot liquid may shatter the loading syringe Accessory Station You must now remove the detergent manually After the cellis empty install the cell cleaning tool Cancel 65 Accessory Station Please connect tubing to the holder This method is used when more vigorous cleaning is required after a precipitating sample or if trouble is suspected It is also a good idea to do this every few weeks or every few days for an instrument under heavy use to prevent problems 66 4 7 Syringe Wash Short Connect the syringe tubing to the syringe fill port Click Syringe Wash Short The software wi
56. s 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 21 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 22 2 2 Experimental Design e TC200 Default User System ITC Options Help Thermostatting ITC 25 T Time Left sy Load Run File Save Run File Experimental Design Advanced Experimental Design Heal Time Plot medal 1 Experimental Hode Results Highest Quality Help Total Heat p Cal e Cell ub Click Calculate button D hs Change 250 _ Help 2 Starting Values Syringe pl Change 2500 Help C Value Estimated Values i TEM i Help gt Kd gooo005 M Help AH 42 kcalsz Mole Help delta H kc alm oie r Legend Within Optimal Range Temperature 25 Degrees C Data File Name Fi O default itc Outside Optimal Range Outside Suggested Range Options __ Use Ka 1 M r Plot gt H 1 0 Molar Ratio When the software 1s first started by default the Experimental Design tab is selected this c
57. s the cause of many problems experienced with the 1T C oo The reference cell generally requires no special cleaning rinsing and refilling every week or so is 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 1s available After every few runs or after any sample that precipitates the sample cell should be cleaned using the Washing Module 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 In the Instrument Controls tab click Cell Buffer Rinse Short or Cell Water Rinse Long if more vigorous cleaning is desired The Washing Module will rinse the cell with buffer and water and remove the liquid If this cleaning method is insufficient use the Detergent Soak and Rinse Long button and follow the prompts to load 5 Contrad70 The cells will heat to 50 C for half an hour to soak Once they have cooled the software will prompt the user to remove the Contrad70 and insert the cleaning device for rinsing Be sure that the cells have cooled before removing the Contrad70 as hot liquid may shatter the loading syringe Syringe Cleaning See Section 4 6 for standard cleaning with the Washing Module If clogging of the syringe 1s suspected it should be cleaned by running the thin wire through it This must be d
58. s the initial setup file which will be loaded into the experimental control window when it is first opened Specifying the initial setup file as LastRun1 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 1TC200 instrument is powered up To save equilibration time set this temperature to the same as the common experimental run temperature 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 uCal sec The Extended Data Mode button will tell the software to save all data the 1T Coo 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 f
59. t gt Apply To Rest Stiring Speed 0 1500 RPM 1000 e Ed 4 48 180 4 101 Feedback Mode Gain 180 lt gt None lt gt Low gt High A AR 30 180 180 180 il 180 1 i dJ A AB 180 Kd 0 000005 dH 42 i 180 i i um 140 HI MDH 180 2 24 A AB 180 j 180 il Data File Name 0 defaudtilc o ilc Data File Comments Mormalized 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 1tc the run parameters are extracted from the data file header or a setup file 1nj which was previously saved using the Save Run File button Save Run File displays the file save 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 25 Display Run Parameters displays the current run parameters for the run in progress This button is available only when the 1TC oo 1s in a non idle state Update Run Parameters updates the current run parameters for the run in progress When the ITC 1s in a non idle state this button must
60. ta 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 1s roughly linearly proportional to each concentration and the injection size The refined estimate of Kg allows for an adjustment of the cell and syringe concentrations As an example assume we have an enzyme in the cell with a Kg of 0 000001 The concentration of enzyme should therefore be 10 Ka 0 00001 M or 10 uM Since this is a reasonably average Kg no further adjustment is 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 28 1TC200 Default User System ITC Options Help gt Load Run File Save Run File Advanced Experimental Design ITC Thermostat Control Set Point C E s 25 El Set Jacket Temp Pulse Control Set Up Pulse Size 5 gy Duration 300 Pulse On Hun Type Y Experiment Control Excel File Hame 2 4 Instrument Controls Cells Passively Cooling to 25 C gt Instrument Controls Pipette Heady Open Fill Port E Purge ReFill Distance in Up n1 Dn Maintenance 1 Remove Old Tip 2 Install New Tip 3 Mew Tip Installed Auto Postrun Data Analys
61. ted especially after a new pipette 1s installed The ITC gt Pipette Tools menu option will open the following popup El Pipette Setup Initialize Pipette Initialize m Perform automaticaly at ITC200 startup Open Port Calibration 1 Adjust Position Use UP and DOWN buttons to adjust the plunger right above the fill port Distance ustem pom Matric 2 Calibrate Once plunger position is obtained use button below to memorize this setting ni Calibrate Open Fill Port The Initialize Pipette section will make the software run a routine that checks the sensors on the pipette and makes sure that the software knows certain constants of the pipette The checkbox at the bottom of the section will cause the software to run this check every time it Starts The Open Port Calibration section contains the controls for adjusting the position of the plunger when the fill port is open Use the distance slider bar and the up and down buttons to move the plunger tip to just above the fill port in the upper side of the glass syringe bore See the pictures below for correct positioning Once the position is correct click the Calibrate Open Fill Port button to have the software set the current location as the open fill port location Click the Open and Close buttons in the bottom section to check the calibration 48 Good Open Port Position Bad Position Too Low Close Port Position 49 3 6 Y axis Calibration Check It is rec
62. temperature to 30 C 2 Set the Plot Idle Data flag so that the current data 1s displayed 3 When the instrument is thermostatting at the target temperature the DP has settled at a negative stable value and the DT 1s 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 If the error for each 1s 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 52 Section 3 8 Configuring an 1TC 9 Controller for Networking On an 1TC oo controller there are several actions that must be performed for the networking and 1TC5oo control software to work properly First the InitDT watchdog program must be removed and once the computer has been configured for the local domain two folders must be given special permissions Uninstallation 1 Click on the START button on the lower left side of the screen and then on Control Panel 2 In the Control Panel window click on Add or Remove Programs 3 Find InitDTSetup in the program list Click the Remove button by it In the next window click Yes to confirm the removal Special Permissions The 1TC oo software and Origin both need to be able to write data into their own folders In order to allow
63. vacuum will remove any excess titrant Once this has completed the software will prompt the user to disconnect the syringe tubing make sure the cells are filled and move the pipette into the sample cell 61 Accessory Station Make sure the cells are filled Please disconnect the syringe tubing and insert the pipette into the cell port Cancel 62 4 4 Cell Buffer Rinse Short The cell cleaning device must be inserted into the sample cell until it seats firmly Connect the syringe tubing to the vacuum block port at the rear of the Washing Module to prevent leakage Click Cell Buffer Rinse Short The Washing Module will perform a short cleaning routine rinsing the cell with buffer Accessory Station Please connect tubing to the holder The program will rinse the cells with buffer and then with water It will then empty the cell although not all the liquid will be removed The entire sequence takes a little over a minute The user may then remove the excess water and load the cell with sample using the Hamilton loading syringe 63 4 5 Cell Water Rinse Long The cell cleaning apparatus must be inserted into the sample cell until it seats firmly Connect the syringe tubing to the vacuum block port to prevent leakage Click Cell Water Rinse Long The Washing Module will perform a more thorough cleaning routine which takes about 80 seconds Like the Cell Buffer Rinse Short the program will rinse the cell thi

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