User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/19: Difference between revisions
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==Purpose== | ==Purpose== | ||
* To determine enzymatic activity of horseradish peroxidase | * To determine enzymatic activity of horseradish peroxidase via the usage of a luminol chemiluminescence assay. | ||
==Procedure== | ==Procedure== | ||
* Stock solutions were pre-made 24 hours ago before samples were mixed and run under fluorometer. See [[User: | * Stock solutions were pre-made 24 hours ago before samples were mixed and run under fluorometer. See [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/18|2012/09/18]] for details in making stock solutions on concentration of horseradish peroxidase and 4-Idophenol that's used in solutions. | ||
* Luminol stock solution was diluted to 2.5mM and mixed with carbonate buffer. See [[User:Melissa Novy/Notebook/CHEM-571/2012/09/19|Melissa's Notebook]] for details of the dilution and reasoning behind making the luminol stock. | * Luminol stock solution was diluted to 2.5mM and mixed with carbonate buffer. See [[User:Melissa Novy/Notebook/CHEM-571/2012/09/19|Melissa's Notebook]] for details of the dilution and reasoning behind making the luminol stock. | ||
* First sample was mixed with 4-Idophenol, H2O2, luminol, and horseradish peroxidase with the concentration below, respectively, before measurements were taken. For details in concentration of stocks added, see below table: | * First sample was mixed with 4-Idophenol, H2O2, luminol, and horseradish peroxidase with the concentration below, respectively, before measurements were taken. For details in concentration of stocks added, see below table: | ||
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* Samples with varying concentrations of luminol or H2O2 were diluted from those stock solutions. To make 625uM luminol, 50uL of 1.25uM was mixed with 50uL of distilled water. To make 850uM of H2O2, 50uL of 1.7mM H2O2 was mixed with 50uL of distilled water. | * Samples with varying concentrations of luminol or H2O2 were diluted from those stock solutions. To make 625uM luminol, 50uL of 1.25uM was mixed with 50uL of distilled water. To make 850uM of H2O2, 50uL of 1.7mM H2O2 was mixed with 50uL of distilled water. For the final concentration of the substrate in each sample, please refer to [[User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/19|Melissa's Notebook]] for the calculated final concentration of substrate in each sample after mixture. | ||
* Fluorometer was set to wavelength 430nm. All data were collected for a period of ~300 seconds to make sure samples has reached equilibrium and had completed in fluorescing. | * Fluorometer was set to wavelength 430nm. All data were collected for a period of ~300 seconds to make sure samples has reached equilibrium and had completed in fluorescing. | ||
==Notes== | ==Notes== |
Revision as of 20:26, 26 October 2012
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Purpose
Procedure
Notes
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