User:Megan L. Channell/Notebook/Horseradish/2013/10/09

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Objective

Continue work from yesterday, but to observe the ADA kinetics with an inhibitor.

Procedure

Creating the Adenosine Stock

  1. Add 0.1073 g of adenosine to a 10 ml volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a stock solution of 0.04 M.
  2. Add 10.00 μL of the 0.04 M adenosine stock to a 10 mL volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a final solution of 40 μM.

Enzyme Kinetics Measurement

  1. Add 3 mL of 40 μM adenosine solution to the cuvette and 30 μL of inosine.
  2. Start kinetics measurement:
  • 1 ms integration
  • 10 scan average
  • Set "Save the first available scan every" to 15 seconds
  • Set "Stop after this amount of time" to 10 minutes
  • Set "File Type" to Tab Delimited
  • Just before 1 minute, add 1.5 μL of 1 nM EHNA.

Data

Image:Cmj_10.09.13_ADAwithinhibitor.png

Notes

EHNA stock

  1. 5mg EHNA in 1mL DMSO ---> 15.9mM EHNA
  2. (1.9μL)(15.9mM EHNA)=(10mL)(C2). C2 ---> 3μM EHNA

The reaction samples will contain roughly 1nM EHNA.




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