User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/19

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Purpose

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

Procedure

  • Stock solutions were pre-made 24 hours ago before samples were mixed and run under fluorometer. See Melissa's Notebook 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 Melissa's Notebook for details of the dilution and reasoning behind making the luminol stock.
  • Samples were mixed with 4-Idophenol, H2O2, luminol, and horseradish peroxidase, respectively, before measurements were taken. For details in concentration of stocks added, see below table:
  • Fluorometer was set to wavelength 510nm. All data were collected for a period of ~300 seconds to make sure samples has reached equilibrium and had completed in fluorescing.

Notes

  • Fluorescence at 510nm was plotted against time in seconds. Only three samples were run: 1.25mM luminol and 1.7mM H2O2, 625uM luminol and 1.7mM H2O2, and 625uM luminol and 850uM H2O2. Within the samples also added 18mM 4-iodophenol and 2.3uM horseradish peroxidase.


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