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Objectives
- Monitor the kinetics and yield of horseradish peroxidase catalyzed oxidation of luminol
- Measure UV-Vis absorbance of luminol, HRP, and Luminol+HRP+H2O2
- Monitor chemiluminscence of luminol oxidation using stop-flow techniques
- Measure kinetics of reaction using stop-flow techniques and absorbance
UV-vis
Stock solutions prepared before class:
- Luminol: 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8
- Buffer: 5mM Tris pH=8
- HRP: 1.7μM HRP in 50mL 5mM Tris pH=8
- H2O2: 117μL of 30%H2O2 in 50mL 5mM buffer.
Samples Run:
- 1/20 dilution of lumino1
- HRP stock solution
- 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)
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