User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/02/13

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Trial 2 & 3 of ADA Kinetic Assay

  • The agenda for this laboratory period are the completion of trials 2 and 3 of ADA Kinetic Assay and obtain the Km and Vmax of the enzymatic reaction.
  • UV2550 Shimadzu Spectrophotometer was baseline with phosphate buffer. The temperature control of the CPS controller was set at 25°C.
  • Baseline was performed by preparing two cuvettes filled with phosphate buffer, one placed on the blank slot and the other at cell slot 1.
  • After baseline, an assay containing 150 μM adenosine was collected using the UV Probe. Trial 2 was complete by running an assay with the 200 μM adenosine.
  • The concentration of each sample was obtained from the calculations below:

Data Table.JPG

  • During the previous laboratory data collection of 100 μM trial 2, an error was encountered by the opening of the cell chamber. Thus, it was decided to repeat the run for 100 μM.
  • 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