Difference between revisions of "User:Catherine Koenigsknecht/Notebook/Experimental Biological Chemistry/2013/02/06"

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#UVProbe was opened.
##Window > 1. Kinetics
##Methods Icon
###Wavelength: 265nm
###Duration: 300 seconds (5minutes)
#Shimadzu CPS-Controller was set to 25°C.
##wait for the temperature to raise to 25°C
#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.

Revision as of 14:49, 8 May 2013

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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

  • Lineweaver Burke Plot:


  • Concentration Adenosine vs Velocity:


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


Concentration recalculations were taken from Dhea Patel Deleted concentration 2.56 uM point from graphs

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