User:Dhea Patel/Notebook/CHEM 572: ADA&Inhibitor Kinetics/2013/02/05

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Procedure)
(Procedure)
Line 29: Line 29:
*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.   
*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).  
*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 calculated to be
+
**The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).
==Observations==
==Observations==

Revision as of 15:14, 5 February 2013

Project name Main project page
Previous entry      Next entry

Objective

  • to perform ADA kinetics assays using UV-vis.

Calculations

  • 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.

Image: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.

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.
  • 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).

Observations


Personal tools