User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/19

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Objective

To determine enzymatic activity of horseradish peroxidase via the usage of fluorometer under 510nm.

The stock solutions of HRP, H₂O₂, and phenol that were made yesterday were mixed and run under the fluorometer at 510nm.
10mL of 2.5mM Luminol stock was prepared using the following calculations:
- MW luminol: 177.16 g/mol
- Mass luminol: 10 mL × (10^-3 L/mL) × (2.5 × 10^-3 M) × (10^-3 mol/mmol) × 177.16 g/mol = 0.00443 g luminol
- Actual mass luminol: 0.0044 g
- Actual concentration: 0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10^-3 M in H₂O
  - Because the luminol did not completely dissolve in H₂O, 0.08g Na₂CO₃ was added to aid in its dissolution.
    - Ratio: 4g sodium carbonate/500mL solution
  - After the luminol dissolved with the sodium carbonate, 0.48g Na₂HCO₃ was added to the solution to neutralize the pH of the solution to about pH 7.
The first sample was mixed with 4-Iodophenol, H₂O₂, luminol, and HRP. Please refer to Kay's entry for details in initial concentration, final concentration, and volumes added.