User:Dhea Patel/Notebook/CHEM 572: ADA&Inhibitor Kinetics/2013/02/12

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==Objective==
==Objective==
* to run ADA kinetics assay
* to run ADA kinetics assay

Revision as of 14:13, 13 February 2013

Search this Project

Customize your entry pages

Objective

  • to run ADA kinetics assay

Methods

  • Baseline 200-400nm using pH7.4, 0.005M phosphate buffer
  • Temperature control: 25°C
  • Phosphate buffer in reference cell
  • Ran kinetics on 250uM solution (20uL ADA, 600uL 1250uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
    • 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.
    • 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.
    • 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.
    • dAbs was obtained and plotted vs. time in excel
  • The following table describes the volumes and concentrations of the components added to the cuvette.
    • Image:Data_Table.JPG

Graphs

Image:TableT1.JPG

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



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