User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/02/12

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(ADA Kinetic Assay)
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==ADA Kinetic Assay==
 
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*The procedure was taken from [[User:Dhea Patel/Notebook/CHEM 572: ADA&Inhibitor Kinetics/2013/02/12|Dhea Patel's]] notebook entry.
 
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*The assay runs were continued from last week's, [[User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/02/05|02/05/2013]], calculated values.
 
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*The objective of performing the assay is to obtain  the rate of the enzymatic reaction of adenosine into inosine by ADA.
 
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*Baseline 200-400nm using pH 7.4, 0.05M phosphate buffer
 
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*Temperature control: 25°C
 
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*Phosphate buffer in reference cell
 
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*Ran kinetics on 250uM solution (20uL ADA, 600uL 1250 uM adenosine, and 2.38mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
 
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**absorbance exceeded 4.00, so the assay was stopped and a new solution was prepared.
 
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*Ran kinetics on 2.56uM solution (20uL ADA, 600uL 12.8 uM adenosine, and 2.38mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
 
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**dAbs was obtained and plotted vs. time in excel
 
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*Ran kinetics on 6.4uM solution (20uL ADA, 600uL 32 uM adenosine, and 2.38mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
 
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**dAbs was obtained and plotted vs. time in excel
 
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*Ran kinetics on 16uM solution (20uL ADA, 600uL 80 uM adenosine, and 2.38mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
 
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**dAbs was obtained and plotted vs. time in excel
 
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*The following table describes the volumes and concentrations of the components added to the cuvette.
 
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[[Image:Data_Table.JPG|center]]
 
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* Note: Since the 250 μM adenosine assay exhibited a high absorbance, this concentration was replaced with two other adenosine concentrations, 150 and 200 μM.
 
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* The stock solution for 150 μM adenosine was made by diluting 150 μL of 5 mM adenosine into 850 ng/μL of 0.05 M phosphate buffer. The 200 μM adenosine was made by diluting 200 μL of 5 mM adenosine into 800 μL of 0.05 M phosphate buffer.
 
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==Graphs==
 
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[[Image:TableT1.JPG]]
 
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[[Image:Avg_Velocity_T1.JPG]] [[Image:Lineweaver-Burk_Plot_T1.JPG]]
 

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