User:Megan L. Channell/Notebook/Horseradish/2013/10/08

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Objective

Measure and observe ADA kinetics without inhibitor. This work will be the basis of our comparison to ADA-AuNPtunrover studies. From Dr. Hartings.


Procedure

  1. Make a 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
  2. Go to Dr. Hartings lab for enzyme kinetics measurements.
    1. Add 3mL of adenosine solution to the cuvette
    2. Start your kinetics measurement
      1. 1ms integration (on front panel)
      2. 10 scan average (on front panel)
      3. Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
      4. Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
      5. Set "File Type" to Tab Delimited
      6. Give the files a directory and a name
      7. Click accept
      8. Just before 1 minute add 30ul of 0.01u/mL ADA
  1. How we made the 40μM solution of adenosine in buffer
    1. .1063 grams of adenosine were dissolved in a 10mL volumetric flask. Molarity: ~40mM
    2. the solution was mixed to ensure homogenity
    3. 9.98 μL of 40mM solution were diluted up to 10mL. Molarity: 40μM

Data

Cmj 10.09.13 ADAnoinhibitor.png

Notes

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