Difference between revisions of "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 11:13, 13 February 2013

<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">User:Dhea Patel/Notebook/CHEM 572: ADA&Inhibitor Kinetics/2013/02/12</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

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Customize your entry pages <html><img src="/images/a/aa/Help.png" border="0" /></html>

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.
    • Data Table.JPG

Graphs

TableT1.JPG

Avg Velocity T1.JPG Lineweaver-Burk Plot T1.JPG