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

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==Objective==
 
*Complete Activity Assay for ADA
 
==Procedure==
==Procedure==
#UVProbe was opened.
#UVProbe was opened.

Revision as of 14:24, 8 May 2013

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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

Concentration of Adenosine (uM) Average Velocity over 30s (nm/sec)
200 0.00021
100 0.000183333
40 0.00012
16 0.0001
6.4 0.000047
2.56 0.000033


1/[adenosine] (1/uM) 1/[velocity] (sec/nm)
0.005 4761.904762
0.01 5454.545455
0.025 8333.333333
0.0625 10000
0.15625 21276.59574
0.390625 30303.0303


-1/Vmax 6163.1
Vmax 0.000162256 nm/sec
-1/Km -0.093
Km 10.75 uM



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