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

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Current revision (23:53, 6 December 2012) (view source)
(Description)
 
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| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
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| Trial||Luminol||H<sub>2</sub>O<sub>2</sub>||4-Iodophenol||HRP||Water||Carbonate Buffer
+
| Trial||Luminol||H<sub>2</sub>O<sub>2</sub>||4-Iodophenol||HRP||Water||0.833mM Sodium 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
| 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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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] '
TrialLuminolH2O24-IodophenolHRPWater0.833mM Sodium Carbonate Buffer
1454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 9.6 µM1033 µLx
2454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 9.6 µM1033 µLx
3454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 9.6 µM2000 µLx
4454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM66 µL at 0.96 µM2000 µLx
5454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 2.3 µMx1000 µL
6454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM16.5 µL at 2.3 µMx1000 µL
7454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM16.5 µL at 0.96 µMx1000 µL
8454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM16.5 µL at 2.3 µMx2000 µL

Data

Image: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.


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