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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Description)
(Description)
Line 21: Line 21:
==Description==
==Description==
-
*The stock solutions of HRP, H<sub>2</sub>O<sub>2</sub>, and phenol that were made [http://openwetware.org/wiki/User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/18 yesterday] were mixed and run under the fluorometer at 510nm.  
+
*The stock solutions of HRP, H<sub>2</sub>O<sub>2</sub>, and phenol that were made [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/18| yesterday]] were mixed and run under the fluorometer at 510nm.  
*10mL of 2.5mM Luminol stock was prepared using the following calculations:
*10mL of 2.5mM Luminol stock was prepared using the following calculations:
**MW luminol: 177.16 g/mol
**MW luminol: 177.16 g/mol

Revision as of 13:30, 2 October 2012

Search this Project

Customize your entry pages

Objective

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

Description

  • 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.

Data

  • Please refer to Kay's entry for the graph produced from the data obtained from 3 samples during fluorescence at 510nm over about 100 seconds. The three samples that ran contained 18mM 4-iodophenol and 2.3uM horseradish peroxidase with varying concentrations of luminol and hydrogen peroxide (1.25mM luminol and 1.7mM H2O2, 625uM luminol and 1.7mM H2O2, and 625uM luminol and 850uM H2O2).



Personal tools