User:Eleni N. Kalivas/Notebook/CHEM-571/2013/09/17: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 30: Line 30:
[[Image:ZoomedinnormalizedabsoxidationHRPzem.png|750px]]
[[Image:ZoomedinnormalizedabsoxidationHRPzem.png|750px]]


#Observations
#* There was an initial drop after the addition of 2uL K3[Fe(CN)6]
#*The addition of 2uL of K3[Fe(CN)6] caused the complete oxidation of HRP. This indicates that HRP was mostly oxidized prior to additon of K3[Fe(CN)6].




Revision as of 10:47, 18 September 2013

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Objective

The objective of today's lab was to determine the amount of reagent necessary to fully reduce horseradish peroxidase (HRP). HRP oxidation will be done with potassium ferricyanide K3[Fe(CN)6]. K3[Fe(CN)6], which has a reduction potential of 424mV. HRP reduction will be done with Sodium Dithionite.

  • the goal is to determine the endpoints of the oxidation and reduction of HRP.

HRP Oxidation

HRP Oxidation Protocol

  • Run UV-vis of a blank buffer sample.
  • Prepare a 1mL sample of 10μM HRP in 50Tris, 50Nacl, pH=7.5 buffer. This buffer should be degassed) directly in a clean glass cuvette.
  • Take spectrum of the prepared 10μM HRP sample.
  • Add 2μL of K3[Fe(CN)6] to the prepared sample.
  • Take UV/vis spectrum of the sample.
  • Repeat steps 4-5 until complete oxidation of HRP.

UV/vis Spectra

Figure 1: Normalized Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]

Figure 2: Enlarged Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]

  1. Observations
    • There was an initial drop after the addition of 2uL K3[Fe(CN)6]
    • The addition of 2uL of K3[Fe(CN)6] caused the complete oxidation of HRP. This indicates that HRP was mostly oxidized prior to additon of K3[Fe(CN)6].




Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Eleni_N._Kalivas/Notebook/CHEM-571/2013/09/17&oldid=729652"