User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/04/19

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(Protocol)
Current revision (17:45, 8 May 2013) (view source)
(Protocol for Assay)
 
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==Objective==
 
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* To obtain a spectrum of 40 μM adenosine and inosine
 
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* A run of 40 μM adenosine with 50 μM varying inhibitors were conducted using the UV 2500 Shimadzu spectrophotometer.
 
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* Three runs of each inhibitor were taken; the average of the runs were taken. The standard deviation were calculated and added as error bars.
 
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* Produce histogram
 
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==Procedure for Adenosine and Inosine Spectrum==
 
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* To obtain spectrum of 40 μM adenosine, dilute 600 μL of 200 μM adenosine stock solution in 2400 μL 0.05 M phosphate buffer.
 
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* Stock solution of 200 μM inosine (MW 268.2 g/mol) was prepared. A spectrum was taken using the same dilution mentioned above.
 
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==Protocol for Assay==
 
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* The reagents were added in the following sequence:
 
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** 1.78 mL of phosphate buffer
 
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** 600 μL of 200 μM adenosine (final concentration 50 μM)
 
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* This was allowed to mix through venting and placed on  the chamber for absorbance reading.
 
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** 300 μL of phosphate buffer (or inhibitor dissolved in phosphate buffer)
 
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** 300 μL of dimethyl sulfoxide (DMSO) (or inhibitor dissolved in DMSO)
 
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* The cuvette contents were mixed by venting and placed on the chamber for absorbance reading.
 
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** 20 μL of adenosine deaminase (ADA) was added to catalyze the reaction
 
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* The kinetics of the reaction was taken using the UVProbe software.
 
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*Cuvette Mixture no Inhibitor
 
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[[Image:CuvetteMixtureNoInhibitor.png]]
 
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*Cuvette Mixture with Inhibitor
 
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[[Image:CuvetteSolnInhibitorHisto.png]]
 
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*Inhibitors Ran today:
 
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# 50 μM 3-methylacetylsalicylic acid (ZINC 00001221)(Aldrich S376647-1G)
 
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# 50 μM Methyl (4-hydroxy-3-methoxyphenyl)acetate (Aldrich PH003466-1MG)
 
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# 50 μM [3,4-bis(acetyloxy)benzoic acid] (Aldrich PH00962-150MG)
 
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