User:Carly M. Montanero/Notebook/CHEM-571/2013/10/16: Difference between revisions

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# Dilute the solution to 10 mL with buffer.
# Dilute the solution to 10 mL with buffer.


 
'''Enzyme Kinetics Measurement'''
# Add 3 mL of the luminol-H<sub>2</sub>O<sub>2</sub> solution to a cuvette.
# Start kinetics measurement:
# Start kinetics measurement:
:* 1 ms integration
:* 1 ms integration
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:* Set "Stop after this amount of time" to 10 minutes
:* Set "Stop after this amount of time" to 10 minutes
:* Set "File Type" to Tab Delimited
:* Set "File Type" to Tab Delimited
:* Just before 1 minute, add 1.5 μL of 1 nM EHNA.
:* Just before 1 minute, add 100 μL of 60:1 Au:HRP solution.
# Take 3 mL of the luminol-H<sub>2</sub>O<sub>2</sub> solution and place it into a cuvette.
#
 
 
*Luminol --> final concentration such that A(330)< 1.
*Hydrogen peroxide --> final concentration to be 50x luminol concentration
*HRP --> 60:1 Au:HRP solutions, 100 μL
*Buffer --> add up to 3 mL


In cuvette, luminol, buffer, and hydrogen peroxide
* Start kinetics by adding HRP


*What percent of HRP is still active?
*What percent of HRP is still active?

Revision as of 10:07, 16 October 2013

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Purpose

To test the activity of our HRP-NPs for the catalytic conversion of luminol.

Procedure

Creating the Luminol-H2O2 Solution

  1. Add 1.293 mL of the luminol stock and 2.131 mL of the hydrogen peroxide stock to a 10 mL volumetric flask.
  2. Dilute the solution to 10 mL with buffer.

Enzyme Kinetics Measurement

  1. Add 3 mL of the luminol-H2O2 solution to a cuvette.
  2. Start kinetics measurement:
  • 1 ms integration
  • 10 scan average
  • Set "Save the first available scan every" to 15 seconds
  • Set "Stop after this amount of time" to 10 minutes
  • Set "File Type" to Tab Delimited
  • Just before 1 minute, add 100 μL of 60:1 Au:HRP solution.


  • What percent of HRP is still active?

Control:

  • experiment with BSA-AuNPs and citrate-AuNPs
  • what concentration of AuNPs about the same (use absorption spectra)

Data

A1V1 = A2V2 (0.3)(100 μL)=A2(3 mL) A2 = ?

Notes

  • The buffer was 5.1 mM Tris at pH 8.
  • The stock concentration of luminol was 1.46 mM.
  • The stock concentration of hydrogen peroxide was 44.29 mM.


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