User:Michael F. Nagle/Notebook/Chem 571/2013/02/06

Objective

• Complete Activity Assay for ADA

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.
• Kinetics was run at 265nm using 2.38mL phosphate buffer, 600μL of 500μM stock adenosine, and 20μL ADA (as outlined in the table below).
  • The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).

• 5mM adenosine stock solution was too concentrated and exceeded the capacity of the UV-Vis therefore it was diluted:
  • 150 uM adenosine stock solution was used; (stock) 150uL 5mM stock + 850 uL buffer =750 uM ->use .6mL new stock for ADA assay
  • 200 uM adenosine stock solution was used: (stock) 200 uL 5mM stock+800 uL buffer = 1000uM ->use 0.6mL new stock for ADA assay
  • This table is the volumes of 5mM adenosine and phosphate buffer used to make the new adenosine stock solutions.
    • 
  • This table is the updated table of the volumes and concentrations of solutions that will be added in the cuvette.
    • 

Data