User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/10/24

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Objective

  • to run Fluorescence on Luminol-HRP assays.

Description

Please refer to table for volumes and concentrations of HRP, Luminol, Phenol, Buffer, and hydrogen peroxide used.

' Volume and Stock Concentration Added to Cuvette ' ' ' Volume Added to Cuvette [µL] '
Trial Luminol H2O2 4-Iodophenol HRP Water Carbonate Buffer
1 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 9.6 µM 1033 µL x
2 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 9.6 µM 1033 µL x
3 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 9.6 µM 2000 µL x
4 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 66 µL at 0.96 µM 2000 µL x
5 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 2.3 µM x 1000 µL
6 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 2.3 µM x 1000 µL
7 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 0.96 µM x 1000 µL
8 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 2.3 µM x 2000 µL

Data

Trail 6.png

  • All the trails, except trial 6 had a straight peak, showing do drop off of the fluorescence. Trail 6, containing, 454uL 0.564mM Luminol, 112.7uL 5mM hydrogen peroxide, 16.5uL 0.9uM HRP, 13.2 uL 18mM 4-Iodophenol, and 1000uL Carbonate Buffer, showed a distinct drop off. Tests should be run, keeping the molarity of 0.9uM constant and varying other factors to determine the optimum molar ratio.