User:Catherine Koenigsknecht/Notebook/Experimental Biological Chemistry/2013/02/06

Biomaterials Design Lab Main project page
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Objective

Learn how to maintain an OpenWetWare Notebook.

Description

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

• Lineweaver Burke Plot:

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

Notes

Concentration recalculations were taken from Dhea Patel

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