Team:METU Turkey/Results Discussion/Isothermal Titration Calorimetry (ITC)

<br>I) Sample Preparation

<br>- thaw protein stock (10 min)
<br>- centrifuge protein stock for 5 min @max speed (5 min)
<br>- prepare solutions
  <br>- adding components (5 min)
  <br>- temperature equilibration (10 min)
  <br>- pH adjustment (5 min)
<br>- degassing solutions 10 min
<br>- total: 45 min

<br>Calculations and Controls

<br>- prepare recipies for protein and ligand solution preparations. Also estimate the acid/base addition quantities for pH adjustments. concentrated acid/base additions may denature protein and/or ligand. Therefore pH adjustments will be done at two stages. 
<br>A) buffer level using concentrated acid/base 
<br>B) protein/ligand solution level using less concentrated acid/base

<br>- if using relatively old enzyme stocks or different buffer components, first be sure that the protein is still active.

 
<br>II)Sample loading

<br>Sample Cell Loading
<br>- Wash the hamilton syringe 2 times w/ W1 and 2 times w/ W2
<br>- Unload water in the sample cell. DO NOT leave residual water in the cell.
<br>- Wash the sample cell w/ 5 times W1 and 5 times w/ W2
<br>- Wash the sample cell w/ buffer which will be used in the experiment
<br>- Slowly fill the syringe with macromolecule solution. Purge the trapped air in the needle. Tap the syringe.
<br>- Load the sample cell. (2 bursts after 1.0 ml). DO NOT inject trapped air in the syringe. Take the excess solution in the sample reservoir.

<br>Injection Syringe loading
<br>- Purge the water in the Injection Syringe   (purge-refill without putting the syringe in any solution or water)
<br>- Attach the filling syringe. Open the fill port. Press “Up” once.
<br>- Pass 1-2 ml of air through the syringe to purge residual water in the syringe.
<br>- Wash the injection syringe w/ W3 before change the ligand (btw two experiments)
<br>- Purge buffer in the syringe once
<br>- Put the syringe into ligand solution
<br>- VERY SLOWLY withdraw the plunger of the filling syringe until you see ligand solution exit through the filling port. IMMEDIATELY  press the close fill port.
<br>- Purge-refill while the injection syringe is in the ligand solution
<br>- Insert the injection syringe into the sample cell and press gently to fit.

<br>III) During the Experiment
<br>- Bring syringe and falcon rack to lab. DO NOT leave them in ITC Room
<br>- Record the sample preparation details of ongoing run into the excel file.
<br>- Analyze the data of previous run and record the results into the excel file.
<br>- Check the ongoing run, if signal is not good, stop the experiment

<br>IV) After The Experiment
<br>- Record set / initial / final values of baseline
<br>- Put post-titration mix into post-titration vial.
  <br>- Check and record turbidity
  <br>- Set thermovac temp to experimental temp of the mix. Thermostat mix (half speed stirring) for 10 min, measure <br>and record the pH
<br>- Label and keep the mixture for enzyme recycling. Take a sample of post-titration mix and conduct activity assays of pre and post titration solutions.


<br>Cleaning Sample Cell
<br>- Use W1 and wash sample cell 7 times and use W2 and wash sample cell 7 times. Fill sample reservoir halfway each time. Rinse the syringe once in between each wash

<br>Cleaning Injection Syringe
<br>- Attach the filling syringe. Open the fill port. Press “Up” once.
<br>- Use W3 to pass 1-2 ml dH2O through the injection syringe. Keep the syringe in W3 and close the fill port. Detach the filling syringe.
<br>- Insert the injection syringe into the port.