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

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

Revision as of 16:24, 8 May 2013

Project name Main project page
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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.
      • Image:Screen_Shot_2013-02-05_at_9.01.20_PM.png
    • This table is the updated table of the volumes and concentrations of solutions that will be added in the cuvette.
      • Image:Screen_Shot_2013-02-05_at_9.01.33_PM.png

Data

Concentration of Adenosine (uM) Average Velocity over 30s (nm/sec)
2000.00021
1000.000183333
400.00012
160.0001
6.40.000047
2.560.000033


1/[adenosine] (1/uM) 1/[velocity] (sec/nm)
0.0054761.904762
0.015454.545455
0.0258333.333333
0.062510000
0.1562521276.59574
0.39062530303.0303


  • Image:Graphsmn1.jpg
-1/Vmax6163.1
Vmax0.000162256 nm/sec
-1/Km-0.093
Km10.75 uM



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