User:Dhea Patel/Notebook/CHEM 572: ADA&Inhibitor Kinetics/2013/02/05

Objective

• to perform ADA kinetics assays using UV-vis.

Calculations

• ADA Kinetics were run using the following volumes and concentrations.
  • Note that the table has been adjusted based on the change in concentration of the adenosine stock solution.

• The kinetics was run using UV-vis at 25°C at 265nm for 5 minutes.

Procedure

1. UVProbe was opened.
   1. Window > 1. Kinetics
   2. Methods Icon
      1. Wavelength: 265nm
      2. Duration: 300 seconds (5minutes)
      3. OK
2. Shimadzu CPS-Controller was set to 25°C.
   1. wait for the temperature to raise to 25°C
3. place the sample in the cell and click start.

• Phosphate buffer was used as a blank and was used to create the baseline.
• A spectrum of 1mM adenosine was taken. The absorbance exceeded the capabilities of the spectrometer, so 100μM adenosine (0.6mL 500μM stock adenosine diluted with 100μM by adding 2.4mL buffer) was used as the maximum concentration.
• Once it was determined that 100μM adenosine created a clear spectra, kinetics was run at 265nm using 2.38mL phosphate buffer, 600μL of 500μM stock adenosine, and 20μL ADA (as outlined in the table above).
  • The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).
• Then, an assay was run at 265nm using 2.38mL phosphate buffer, 600μL of 50μM stock adenosine, and 20μL ADA (as outlined in the table above).
  • The absorbance was close to 0, indicating that the ideal adenosine concentration likely ranges between 10μM and 100μM.

Observations