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

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(New page: <br>CLSM IMAGING <br>- Take 10mL from the samples (OD=1-1.9) that are dissolved in phosphate buffer solution. <br>- Spread the samples on the slide with the help of an inoculation loop. ...)
 
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<br>CLSM IMAGING
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<br>I) Sample Preparation
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<br>- Take 10mL from the samples (OD=1-1.9) that are dissolved in phosphate buffer solution.
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<br>- thaw protein stock (10 min)
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<br>- Spread the samples on the slide with the help of an inoculation loop.
+
<br>- centrifuge protein stock for 5 min @max speed (5 min)
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<br>- Wait for the sample to dry.
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<br>- prepare solutions
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<br>- Pass the slide through the flame 3-4 times, being careful to have the cells on the upper part of the slide. 
+
  <br>- adding components (5 min)
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<br>- Observe the slides under Brightfield 100X
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  <br>- temperature equilibration (10 min)
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<br>-Settings for GFP: HFT 488
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  <br>- pH adjustment (5 min)
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                            <br>Ch 1
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<br>- degassing solutions 10 min
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                            <br>LP 505
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<br>- total: 45 min
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                            <br>Line 8
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                            <br>543 nm excitation
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                            <br>Laser power 5%
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<br>- Settings for RFP: HFT 488/543
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<br>Calculations and Controls
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                            <br>Ch 1
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-
                            <br>LP 560
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<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>Line 8
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<br>A) buffer level using concentrated acid/base
-
                            <br>543 nm excitation
+
<br>B) protein/ligand solution level using less concentrated acid/base
-
                            <br>Laser power 10%
+
 
 +
<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.

Latest revision as of 02:45, 28 October 2010


I) Sample Preparation


- thaw protein stock (10 min)
- centrifuge protein stock for 5 min @max speed (5 min)
- prepare solutions 

 
- adding components (5 min)
 
- temperature equilibration (10 min)
 
- pH adjustment (5 min)


- degassing solutions 10 min
- total: 45 min


Calculations and Controls


- 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.
A) buffer level using concentrated acid/base
B) protein/ligand solution level using less concentrated acid/base


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



II)Sample loading


Sample Cell Loading
- Wash the hamilton syringe 2 times w/ W1 and 2 times w/ W2
- Unload water in the sample cell. DO NOT leave residual water in the cell.
- Wash the sample cell w/ 5 times W1 and 5 times w/ W2
- Wash the sample cell w/ buffer which will be used in the experiment
- Slowly fill the syringe with macromolecule solution. Purge the trapped air in the needle. Tap the syringe.
- 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.


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


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


IV) After The Experiment
- Record set / initial / final values of baseline
- Put post-titration mix into post-titration vial. 

 
- Check and record turbidity
 
- Set thermovac temp to experimental temp of the mix. Thermostat mix (half speed stirring) for 10 min, measure 
and record the pH


- 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.



Cleaning Sample Cell
- 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


Cleaning Injection Syringe
- Attach the filling syringe. Open the fill port. Press “Up” once.
- 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.
- Insert the injection syringe into the port.

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