User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/04/19: Difference between revisions
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== | ==Objective== | ||
* | * To obtain a spectrum of 40 μM adenosine and inosine | ||
* A run of 40 μM adenosine with 50 μM varying inhibitors were conducted using the UV 2500 Shimadzu spectrophotometer. | |||
* Three runs of each inhibitor were taken; the average of the runs were taken. The standard deviation were calculated and added as error bars. | |||
* Produce histogram | |||
==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== | |||
* 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 | |||
[[Image:CuvetteMixtureNoInhibitor.png]] | |||
*Cuvette Mixture with Inhibitor | |||
[[Image:CuvetteSolnInhibitorHisto.png]] | |||
*Inhibitors Ran today: | |||
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Revision as of 21:54, 18 April 2013
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Objective
Procedure for Adenosine and Inosine Spectrum
Protocol
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