Isothermal Titration Calorimeter 1. If you have no idea of your
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1. 13 14 15 16 To load your ligand into the ITC syringe first put the tubing of the loading syringe onto the silver filling port Click Open Fill Port on the lower right of the ITC Controls window Place paddle in the tube of your degassed ligand with the tip almost at the bottom Pull on the loading syringe to draw your ligand up into the ITC syringe When the syringe is completely full and some ligand has come out into the loading tubing click Close Fill Port If there is a bubble at the very top of the ITC syringe ignore it even if it seems large MicroCal has shown that a bubble just below the plunger tip does not affect injection accuracy If you think there s a chance that bubbles may have been drawn into the ITC syringe along with your ligand remove the loading syringe tubing and run the Purge Refill function once or twice to remove them If there were any you ll see them appear in the ligand tube To remove air from the bottom of the paddle set the Distance in the pipette control window to something tiny like 0 01 in Remove the ligand tube from the injector assembly then click Down to expel a tiny amount of liquid onto a Kimwipe Do that twice Carefully insert the pipette into the sample cell access tube the hole on the right Near the bottom you have to push a little against a rubber O ring to seat the assembly Be sure the toothed wheel is right next to the stirrer platform You can wait until the machine reac2. auto machine will proceed on its own through equil steps Set Injection Parameters as follows Volume 5 ul maybe see special injection notes at the bottom of this SOP Duration 10 sec default is always 2X volume to allow time for return to baseline Spacing between injections 240 typical range 180 300 Filter period 2 higher for slow thermal process Edit Mode Can make different settings for first injection with Unique button 7 Ifit has been over a week since degassed water was put in the reference cell load the reference cell with 1 8 ml of degassed water Place the needle all the way at the bottom of the chamber then withdraw it back up 1mm and leave it in place as you slowly add the water i e don t withdraw the needle as you deliver water Once the water comes up into the tube stir the water in the chamber as much as you can with the needle to dislodge any air bubbles Then remove the two air bubbles from the shoulders of the chamber using quick pressure to flush them out and once they are gone remove excess volume from the cell by placing the delivery needle on the cell s inner ledge drawing fluid out until you pull air out Ledge Formed where the cell tube meets the plastic filling port Position where bubbles may rest Sample reservoir 8 After rinsing the sample cell twice with 2 ml of your buffer load 1 8 ml of your protein sample into the sample cell as in Step 7 10 11 12
3. Isothermal Titration Calorimeter If you have no idea of your system s binding constant suggestions for starting concentrations of protein and ligand solutions are given in Section 6 5 of the VP ITC User s Manual available online at www microcalorimetry com Protein and ligand solutions need to be in the same buffer so no heat of dilution will be generated when they mix If possible dialyze protein and ligand solutions against the buffer they re in and use the dialysis buffer in the reference cell The Sample Preparation Tips section of the ITC s webpage discusses several parameters DTT and TCEP are better avoided in your buffer See Section 6 3 of the User s Manual for reasons and possible substitutes Turn on the computer first Log on as ITC User using the password itc Then turn on the instrument itself Switch is a rocking toggle located at the upper left on the back Open the VPViewer 2000 software at least 30 seconds after turning on the instrument Otherwise there won t be a Y axis scale chosen under the Setup Maintenance tab or constants loaded under the Constants tab Finally turn on power to the Thermo Vac Switch is a rocking toggle at the upper right on the back Solutions need to be degassed first since air coming out of solution will also introduce spurious heat Set the temperature of the Thermo Vac unit to 3 4 below your experimental run temp since it s much quicker for the machine to heat the sample a little than
4. hes ITC Pre run to insert the injector if you prefer but that might cause the temp to stay 1 higher than the set temp Click on the Start flag to start the run Check on the Display window to see that the Jacket and Sample temperatures have reached the desired levels When they have the red status letters change from Heating to ITC Pre run The machine will then go into Pre stirring equilibration mode If it stays there for 20 30 minutes hit Stop then Start again quickly and it will cycle through all its equilibrations but faster this time When it reaches Final baseline equilibration the stirrer will start If at this point the lower right window pipette control goes gray it means the syringe is not communicating so check the wire connection between the syringe and the box You may need to wiggle that to make the connection and start again The next stage will be ITC Pre titration Delay You can click on the Rescale to Show All button to see your whole trace Finally you will get to ITC injection 1 and the experiment is off and running You can calculate the approximate running time by multiplying your number of injections times the spacing time interval If you think you have enough injections you can stop the ITC at any time by clicking on the STOP sign on the ITC Controls tab You can also let it finish all 17 18 19 the injections you entered in the Method or let it keep injecting until it runs out of
5. ligand To analyze your data click on the MicroCal ITC Analysis icon This opens a second version of Origin not interlaced with instrument control Follow the instructions in the Analyzing ITC Data protocol on the website To clean up the machine for the next user remove your sample and rinse the sample chamber with water Then hook up the automatic washing system and put the injector in the loop so that detergent solution will pass through it as well as the sample chamber Pull 200 250 ml of 5 Liqui Nox through everything followed by an equal volume of water Leave water in the sample chamber Rinse out the injector with methanol by hand then pull air through it for 10 15 minutes using the vacuum set up Leave the paddle in air To close the VPViewer software pull down from System and choose Quit Program Closing the window from the X in the upper right hand corner will result in your getting stuck in an infinite loop Once the software is closed you can turn off the ITC with the switch on the back Leave the computer on Injection notes Set your first injection to be 0 5 ul This will expel the tiny bubble that forms at the tip of the syringe when you insert the syringe into the sample cell This way your first full size injection the second one will be more accurate Delete the data from the first tiny injection before doing curve fitting in Origin You should note how long it takes for the baseline to be reached again after the fir
6. st tiny injection and program future runs to include that delay Updated 5 21 07 Note This protocol is a practical hands on guide to using the instrument Reading this is not a substitute for reading the manufacturer s manual something we strongly recommend that you do
7. to cool it Note there is a missing decimal point on the temperature display 24 looks like 240 Put 2 5 ml of protein solution in a plastic vial with a stir bar and put 500ul of ligand solution in a small glass tube Both can be degassed at the same time Place the vacuum cap over the vacuum chamber and tighten the silver bleeder valve on top of it to increase vacuum Watch for foaming of both solutions at first and reduce vacuum by turning the bleeder valve counterclockwise to calm that as necessary Standard degassing lasts 8 minutes flip vacuum switch to Timer but you can vary that by flipping the vacuum switch to On and timing the degassing yourself While things are degassing open the Thermostat Calibration window Set temperature here at the actual run temperature you want Adjust temperature with up and down arrows Click Set Jacket Temp to set it The instrument s temperature range is 2 to 80 Open ITC Controls window Set Experimental Parameters as follows Total injections up to 500 Cell temperature actual temp you want Reference Power 15 middle of the range Initial delay at least 60 sec Syringe conc conc of ligand can add this later Cell conc conc of protein can add this later Stirring speed 310 is recommended suspended particles will require higher speed Data file name your choice itc unique characters in first ten Feedback Mode high for faster response times ITC Equil Options
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