User:Michael F. Nagle/Notebook/Chem 571/2013/02/06

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==Entry title==
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==Objective==
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* Insert content here...
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*Complete Activity Assay for ADA
 +
==Procedure==
 +
#UVProbe was opened.
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##Window > 1. Kinetics
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##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]]
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**This table is the updated table of the volumes and concentrations of solutions that will be added in the cuvette.
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***[[Image:Screen_Shot_2013-02-05_at_9.01.33_PM.png]]
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==Data==
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Concentration of Adenosine (uM)'''
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| align="center" style="background:#f0f0f0;"|'''Average Velocity over 30s (nm/sec)'''
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|-
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| 200||0.00021
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|-
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| 100||0.000183333
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|-
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| 40||0.00012
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|-
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| 16||0.0001
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|-
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| 6.4||0.000047
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|-
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| 2.56||0.000033
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|}
 +
 
 +
 
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{| {{table}}
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| 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
 +
|-
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| 0.15625||21276.59574
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|-
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| 0.390625||30303.0303
 +
|}
 +
 
 +
 
 +
*[[Image:Graphsmn1.jpg]]
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 +
{| {{table}}
 +
| -1/Vmax||6163.1
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|-
 +
| '''Vmax'''||'''0.000162256 nm/sec'''
 +
|-
 +
| -1/Km||-0.093
 +
|-
 +
| '''Km'''||'''10.75 uM'''
 +
|}
 +
 

Revision as of 00:58, 12 February 2013

Project name Main project page
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Objective

  • Complete Activity Assay for ADA

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.
  • 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

Concentration of Adenosine (uM) Average Velocity over 30s (nm/sec)
2000.00021
1000.000183333
400.00012
160.0001
6.40.000047
2.560.000033


1/[adenosine] (1/uM) 1/[velocity] (sec/nm)
0.0054761.904762
0.015454.545455
0.0258333.333333
0.062510000
0.1562521276.59574
0.39062530303.0303


  • Image:Graphsmn1.jpg
-1/Vmax6163.1
Vmax0.000162256 nm/sec
-1/Km-0.093
Km10.75 uM



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