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- UVProbe was opened.
- Window > 1. Kinetics
- Methods Icon
- Wavelength: 265nm
- Duration: 300 seconds (5minutes)
- Shimadzu CPS-Controller was set to 25°C.
- wait for the temperature to raise to 25°C
- 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.
|Concentration of Adenosine (uM)
||Average Velocity over 30s (nm/sec)
- Concentration Adenosine vs Velocity:
| Vmax||0.000162256 nm/sec
| Km||10.75 uM
Concentration recalculations were taken from Dhea Patel
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