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.
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| Volume and Stock Concentration Added to Cuvette
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| Volume Added to Cuvette [µL]
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| Trial | Luminol | H2O2 | 4-Iodophenol | HRP | Water | 0.833mM Sodium Carbonate Buffer
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| 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
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| 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
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| 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
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| 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
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| 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
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| 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
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| 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
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| 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
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Data
- 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.
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