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

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==ADA Kinetics Assay==
==ADA Kinetics Assay==
-
* The spectrophotometer used was the make of Shimadzu UV-2550 connected to a Shimadzu CPS-temperature controller.
+
* The objective of executing adenosine deaminase (ADA) kinetic assay is to obtain the slope or rate of the enzymatic reaction of the conversion of adenosine into inosine by ADA.
-
* On the UV Probe system interface, the kinetics program was chosen under the Tools option. The parameters were adjusted from the method button.
+
*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 [[User:Dhea Patel/Notebook/CHEM 572: ADA&Inhibitor Kinetics/2013/02/05|Dhea Patel's notebook]].
 +
 
 +
==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==
 +
#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.
 +
*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.
 +
***[[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)'''
 +
|-
 +
| 100||0.000345161
 +
|-
 +
| 10||0.000109677
 +
|-
 +
| 1||2.58065E-05
 +
|}
 +
 
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''1/[adenosine] (1/uM)'''
 +
| align="center" style="background:#f0f0f0;"|'''1/[velocity] (sec/nm)'''
 +
|-
 +
| 0.01||2897.196262
 +
|-
 +
| 0.1||9117.647059
 +
|-
 +
| 1||38750
 +
|}
 +
 
 +
 
 +
 
 +
Lineweaver-Burk Plot of the data obtained today.
 +
[[Image:Lineweaver-Burk_Plot_2013-02-05.png]]
 +
 
 +
{| {{table}}
 +
| 1/Vmax||4015.59923
 +
|-
 +
| '''Vmax'''||'''0.000249029 nm/sec'''
 +
|-
 +
| 1/Km||0.111964094
 +
|-
 +
| '''Km'''||'''8.93143474 uM'''
 +
|}

Revision as of 11:51, 8 February 2013

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ADA Kinetics Assay

  • The objective of executing adenosine deaminase (ADA) kinetic assay is to obtain the slope or rate of the enzymatic reaction of the conversion of adenosine into inosine by ADA.
  • 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.

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).
  • 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.
      • 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

Concentration of Adenosine (uM) Average Velocity over 30s (nm/sec)
1000.000345161
100.000109677
12.58065E-05
1/[adenosine] (1/uM) 1/[velocity] (sec/nm)
0.012897.196262
0.19117.647059
138750


Lineweaver-Burk Plot of the data obtained today. Image:Lineweaver-Burk_Plot_2013-02-05.png

1/Vmax4015.59923
Vmax0.000249029 nm/sec
1/Km0.111964094
Km8.93143474 uM



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