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[[Image:HRP diluteluminol h2o2 zem.png]]
[[Image:HRP diluteluminol h2o2 zem.png]]
==Kinetics of luminol oxidized by HRP==
General Protocol:

Revision as of 12:29, 1 October 2013

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  1. Monitor the kinetics and yield of horseradish peroxidase catalyzed oxidation of luminol
    1. Measure UV-Vis absorbance of luminol, HRP, and Luminol+HRP+H2O2
    2. Monitor chemiluminscence of luminol oxidation using stop-flow techniques
    3. Measure kinetics of reaction using stop-flow techniques and absorbance


Stock solutions prepared before class:

  1. Luminol: 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8
  2. Buffer: 5mM Tris pH=8
  3. HRP: 1.7μM HRP in 50mL 5mM Tris pH=8
  4. H2O2: 117μL of 30%H2O2 in 50mL 5mM buffer.

Samples Run:

  1. 1/20 dilution of lumino1
  2. HRP stock solution
  3. Reaction of HRP+Luminol stock+H2O2 (prepared solution by taking 1/3mL of each sample and allowing to react for ~5min)

Sample notes:

  • Performed 1/20 dilution of 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8. (The luminol stock solution).
    • Make a 10mL stock solution of 1/20 dilute luminol stock solution.
  • Prepared stock solution of hydrogen peroxide was too dilute to measure (characteristic peak at 432 nm was not present)

Figure 1. Corrected Absorbance Spectra of preliminary kinetics
HRP diluteluminol h2o2 zem.png

Kinetics of luminol oxidized by HRP

General Protocol: