Measure and observe ADA kinetics without inhibitor. This work will be the basis of our comparison to ADA-AuNPtunrover studies.
From Dr. Hartings.
- Make a 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
- Go to Dr. Hartings lab for enzyme kinetics measurements.
- Add 3mL of adenosine solution to the cuvette
- Start your kinetics measurement
- 1ms integration (on front panel)
- 10 scan average (on front panel)
- Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
- Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
- Set "File Type" to Tab Delimited
- Give the files a directory and a name
- Click accept
- Just before 1 minute add 30ul of 0.01u/mL ADA
- How we made the 40μM solution of adenosine in buffer
- .1063 grams of adenosine were dissolved in a 10mL volumetric flask. Molarity: ~40mM
- the solution was mixed to ensure homogenity
- 9.98 μL of 40mM solution were diluted up to 10mL. Molarity: 40μM
Adenosine deaminase (ADA) stock
- 1.1mg (24.0 units in 1 mg) in 25mL of buffer --> 1.1 units/mL ADA
ADA for experiments
- 100uL of stock ADA and 900uL buffer --> 0.11 units/mL ADA