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

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(ADA Kinetic Assay)
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*Temperature control: 25°C
*Temperature control: 25°C
*Phosphate buffer in reference cell
*Phosphate buffer in reference cell
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*Ran kinetics on 250uM solution (20uL ADA, 600uL 1250uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
+
*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.
**absorbance exceeded 4.00, so the assay was stopped and a new solution was prepared.  
**absorbance exceeded 4.00, so the assay was stopped and a new solution was prepared.  
-
*Ran kinetics on 2.56uM solution (20uL ADA, 600uL 12.8uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
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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.
**dAbs was obtained and plotted vs. time in excel
**dAbs was obtained and plotted vs. time in excel
-
*Ran kinetics on 6.4uM solution (20uL ADA, 600uL 32uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
+
*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.
**dAbs was obtained and plotted vs. time in excel
**dAbs was obtained and plotted vs. time in excel
-
*Ran kinetics on 16uM solution (20uL ADA, 600uL 80uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
+
*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.
**dAbs was obtained and plotted vs. time in excel
**dAbs was obtained and plotted vs. time in excel
*The following table describes the volumes and concentrations of the components added to the cuvette.  
*The following table describes the volumes and concentrations of the components added to the cuvette.  
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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.
* Note: Since the 250 μM adenosine assay exhibited a high absorbance, this concentration was replaced with two other adenosine concentrations, 150 and 200 μM.
* 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.
* 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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Revision as of 15:51, 13 February 2013

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ADA Kinetic Assay

  • The procedure was taken from Dhea Patel's notebook entry.
  • The assay runs were continued from last week's, 02/05/2013, calculated values.
  • The objective of performing the assay is to obtain the rate of the enzymatic reaction of adenosine into inosine by ADA.
  • Baseline 200-400nm using pH 7.4, 0.05M phosphate buffer
  • Temperature control: 25°C
  • Phosphate buffer in reference cell
  • 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.
    • absorbance exceeded 4.00, so the assay was stopped and a new solution was prepared.
  • 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.
    • dAbs was obtained and plotted vs. time in excel
  • 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.
    • dAbs was obtained and plotted vs. time in excel
  • 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.
    • dAbs was obtained and plotted vs. time in excel
  • The following table describes the volumes and concentrations of the components added to the cuvette.



  • Note: Since the 250 μM adenosine assay exhibited a high absorbance, this concentration was replaced with two other adenosine concentrations, 150 and 200 μM.
  • 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.

Graphs

Image:TableT1.JPG

Image:Avg_Velocity_T1.JPG Image:Lineweaver-Burk_Plot_T1.JPG



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