Difference between revisions of "User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/04/19"

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(Protocol for Assay)
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==Procedure for Adenosine and Inosine Spectrum==
* To obtain spectrum of 40 μM adenosine, dilute 600 μL of 200 μM adenosine stock solution in 2400 μL 0.05 M phosphate buffer.
* Stock solution of 200 μM inosine (MW 268.2 g/mol) was prepared. A spectrum was taken using the same dilution mentioned above.
==Protocol for Assay==
* The reagents were added in the following sequence:
** 1.78 mL of phosphate buffer
** 600 μL of 200 μM adenosine (final concentration 50 μM)
* This was allowed to mix through venting and placed on  the chamber for absorbance reading.
** 300 μL of phosphate buffer (or inhibitor dissolved in phosphate buffer)
** 300 μL of dimethyl sulfoxide (DMSO) (or inhibitor dissolved in DMSO)
* The cuvette contents were mixed by venting and placed on the chamber for absorbance reading.
** 20 μL of adenosine deaminase (ADA) was added to catalyze the reaction
* The kinetics of the reaction was taken using the UVProbe software.
*Cuvette Mixture no Inhibitor
*Cuvette Mixture with Inhibitor
*Inhibitors Ran today:
# 50 μM 3-methylacetylsalicylic acid (ZINC 00001221)(Aldrich S376647-1G)
# 50 μM Methyl (4-hydroxy-3-methoxyphenyl)acetate (Aldrich PH003466-1MG)
# 50 μM [3,4-bis(acetyloxy)benzoic acid] (Aldrich PH00962-150MG)
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Revision as of 15:45, 8 May 2013

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