User:Michael F. Nagle/Notebook/Chem 571/2013/02/06: Difference between revisions
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== | ==Objective== | ||
* | *Complete Activity Assay for ADA | ||
==Procedure== | |||
#UVProbe was opened. | |||
##Window > 1. Kinetics | |||
##Methods Icon | |||
###Wavelength: 265nm | |||
###Duration: 300 seconds (5minutes) | |||
###OK | |||
#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. | |||
***[[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== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Concentration of Adenosine (uM)''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''1/[adenosine] (1/uM)''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} | |||
*[[Image:Graphsmn1.jpg]] | |||
{| {{table}} | |||
| -1/Vmax||6163.1 | |||
|- | |||
| '''Vmax'''||'''0.000162256 nm/sec''' | |||
|- | |||
| -1/Km||-0.093 | |||
|- | |||
| '''Km'''||'''10.75 uM''' | |||
|} | |||
Revision as of 21:58, 11 February 2013
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Objective
Procedure
Data
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