CMI ITC-200 Standard Protocol - Center for Macromolecular
Contents
1. CENTER FOR MACROMOLECULAR INTERACTIONS MicroCal ITC 200 for Isothermal Titration Calorimetry General Care and Maintenance e The instrument should be left ON at all times e Wash the instrument thoroughly before and after every run e Treat the syringe with care it s easy to break and expensive e Report problems immediately in the booking system https ppms us hms cmi e Refer to the Microcal ITC 200 Getting Started Guide and User Manual for more information e Perform water in water titrations at the end of the day to check the system Start up Sequence Before you start book time on the PPMS calendar oF Login to the computer using your PPMS credentials eCommons ID and password The ITC 200 instrument should be left ON at all times If not turn it on using the power switch on the back Start the MicroCal ITC 200 software After system initialization check that all systems are working and that the red light on the front of the instrument is on Make sure that wash station bottles and methanol are at least full a Methanol bottle should contain 100 Methanol b Water and Buffer bottles should both contain Milli Q water Empty the waste bottle if more than full Fill the reference cell with water if it hasn t been filled in more than a week In ITC 200 software select Instrument Controls Tab 10 Run Cell Water Rinse long 11 Run Syringe Wash long a follow on screen directions for attaching the fill port adap2. book Logoff from PPMS rates are based on booked and real time usage Report Problems in the PPMS booking system https ppms us hms cmi h Contact cmi hms harvard edu with questions last edited 2015 10 30 CMI e Harvard Medical School e 45 Shattuck St e LHRRB 211A e Boston MA 02115 cmi hms harvard edu 617 432 5004 e Kelly Arnett PhD Director
3. lly fill cell with cell sample being very careful not to introduce air bubbles fill Hamilton syringe with about 300 ul sample for cell remove air bubbles carefully insert syringe into sample cell until it touches the bottom lift syringe up 1 2 mm slowly fill cell with sample until sample emerges from top gently aspirate and dispense about 50 ul sample to dislodge any air bubbles f remove the syringe while ejecting g place the tip of the syringe on the ledge at top of metal cell stem and remove excess solution 10 When syringe fill is complete disconnect the fill port adapter a move pipette to rest position b loosen retaining nut c remove fill port adapter d gently tighten retaining nut 11 Place pipette into cell port 12 Click start to begin assay a in 5 10 min the differential power DP should stabilize near the set reference power 1 ucal sec b if it doesn t stabilize at all or near the reference power check that the sample cell and the reference cell are filled or clean the cell again 13 Select Real Time Plot to view raw data 14 After every run clean the cell and syringe again before the next titration 15 At the end of the day follow the NEW Shutdown Procedure below and posted at the instrument angop CMI e Harvard Medical School e 45 Shattuck St e LHRRB 211A e Boston MA 02115 cmi hms harvard edu 617 432 5004 e Kelly Arnett PhD Director CENTER FOR MACROMOLECULAR INTERACTIONS New ITC 200 Shutdown P
4. rocedure 1 N O Oie Clean the instrument a Clean the cell using the Cell Water Rinse long b Clean the syringe using the Syringe Wash Long protocol c If you suspect precipitation in the cell wash the cell with detergent i follow the Detergent Soak and Rinse protocol with 10 20 Contrad 70 ii Remove the detergent manually from the cell using the Hamilton syringe iii Wash the cell using the wash module Run a water in water titration a Fill the syringe with filtered degassed water b Fill the cell with filtered degassed water c Detach the wash module from the syringe d In ITC 200 software go to Setup Tab i enter Data File path C ITC200 Data 1_CMI_WaterTitrations ii enter Setup File Path C ITC200 Setup 1_CMI_WaterTitrations e In ITC 200 software select Advanced Experimental Design Tab Load the file Water_15cycle_RP6 inj or set up the experiment with the following parameters i 15 injections 25C Reference Power 6ucal sec Initial Delay 60 sec ii Data File Name YYYYMMDDwaterINITIALS eg 2015051 9waterKLA iii 2 ul injections 120 sec spacings Start the water in water titration runtime 30 min During the run check that i the differential power is within 1ucal sec of the set reference power ii Check that the heats of dilution are minimal and uniform iii If these fails repeat from Step 1 until the instrument is clean Run the Syringe Wash Long or Short protocol Empty the cell Sign in the paper log
5. ter b do not over tighten fill port adapter Standard Protocol 1 Prepare samples and reagents see CMI_ITC200_sample_prep_ guidelines document for tips 2 In ITC 200 software select Setup Tab a enter Data File path eg C ITC200 Data PlnameLab YourName b enter Setup File Path optional c Current User should be Default User do not create user profiles 3 In ITC 200 software select Advanced Experimental Design Tab 4 Enter Experimental Parameters Total Injections 19 Cell Temperature C 25 Reference Power ucal s 6 7 if AH is unknown Initial Delay sec 60 CMI e Harvard Medical School e 45 Shattuck St e LHRRB 211A e Boston MA 02115 cmi hms harvard edu 617 432 5004 e Kelly Arnett PhD Director CENTER FOR MACROMOLECULAR INTERACTIONS Syringe concentration mM Cell concentration mM Stirring Speed RPM 1000 Data File Name Feedback Mode Gain high 5 Enter Injection Parameters first inj remaining injections Volume ul 0 4 2 Duration sec 0 8 4 2X injection volume Spacing sec 150 150 Filter Period sec 5 5 6 In ITC 200 software select Instrument Controls Tab 7 Run Syringe Fill Method a place a tube with 260 ul syringe sample in the tube holder at load position b follow on screen directions for filling syringe c Don t forget to remove the wash module attachment after filling 8 Manually wash cell with buffer matched to sample buffer at least 3 times using Hamilton syringe 9 Manua
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