User:Carly M. Montanero/Notebook/CHEM-571/2013/10/09: Difference between revisions

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==Figures==
==Figures==


==Data==
==Notes==
'''EHNA stock'''
'''EHNA stock'''
# 5mg EHNA in 1mL DMSO ---> 15.9mM EHNA
# 5mg EHNA in 1mL DMSO ---> 15.9mM EHNA
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The reaction samples will contain roughly 1nM EHNA.
The reaction samples will contain roughly 1nM EHNA.


==Notes==
==Notes==

Revision as of 09:29, 9 October 2013

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Objective

To observe and measure ADA turnover kinetics in the presence of an inhibitor, EHNA. This work will be the basis of our comparison to ADA-AuNP turnover studies.

From Dr. Hartings.

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.
  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 μL of 1 nM EHNA.

Figures

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.

Notes

  • The stock solution of HRP was 0.77 μM.
  • The stock solution of luminol was 1.51 mM.
  • The buffer solution was 5.1 mM Tris at pH = 8.
  • The stock solution of hydrogen peroxide was 12.8 mM.



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