User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/02/05

From OpenWetWare
< User:Mary Mendoza‎ | Notebook‎ | CHEM572 Exp. Biological Chemistry II‎ | 2013‎ | 02
Revision as of 21:58, 7 February 2013 by Mary Mendoza (talk | contribs) (ADA Kinetics Assay)
Jump to: navigation, search
Owwnotebook icon.png Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

ADA Kinetics Assay

  • The spectrophotometer used was the make of Shimadzu UV-2550 connected to a Shimadzu CPS-temperature controller.
  • On the UV Probe system interface, the kinetics program was chosen under the Windows option. The parameters were adjusted from the method button.
  • The rest of the procedure is taken from Dhea Patel's notebook.


  • ADA Kinetics were run using the following volumes and concentrations.
    • Note that the table has been adjusted based on the change in concentration of the adenosine stock solution.

Screen Shot 2013-02-05 at 11.59.42 AM.png

  • The kinetics was run using UV-vis at 25°C at 265nm for 5 minutes.


  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.
  • A spectrum of 1mM adenosine was taken. The absorbance exceeded the capabilities of the spectrometer, so 100μM adenosine (0.6mL 500μM stock adenosine diluted with 100μM by adding 2.4mL buffer) was used as the maximum concentration.
  • Once it was determined that 100μM adenosine created a clear spectra, kinetics was run at 265nm using 2.38mL phosphate buffer, 600μL of 500μM stock adenosine, and 20μL ADA (as outlined in the table above).
    • The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).
  • Then, an assay was run at 265nm using 2.38mL phosphate buffer, 600μL of 50μM stock adenosine (to make a final concentration of 10μM), and 20μL ADA (as outlined in the table above).
    • The absorbance was close to 0, indicating that the ideal adenosine concentration likely ranges between 10μM and 100μM.
  • Then, an assay was run at 265nm using 2.38mL phosphate buffer, 600μL of 5μM stock adenosine (to make a final concentration of 1μM), and 20μL ADA (as outlined in the table above).
  • From the spectra obtained today, the following tables were created:
    • This table is the volumes of 5mM adenosine and phosphate buffer used to make the new adenosine stock solutions.
      • 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.
      • Screen Shot 2013-02-05 at 9.01.33 PM.png


Concentration of Adenosine (uM) Average Velocity over 30s (nm/sec)
100 0.000345161
10 0.000109677
1 2.58065E-05
1/[adenosine] (1/uM) 1/[velocity] (sec/nm)
0.01 2897.196262
0.1 9117.647059
1 38750

Lineweaver-Burk Plot of the data obtained today. Lineweaver-Burk Plot 2013-02-05.png

1/Vmax 4015.59923
Vmax 0.000249029 nm/sec
1/Km 0.111964094
Km 8.93143474 uM