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

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(UV/vis Spectra)
(HRP Reduction)
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=HRP Reduction=
 
=HRP Reduction=
 +
'''HRP Reduction Protocol'''
 +
#Run UV-vis of a blank buffer sample.
 +
#Prepare a 1mL sample of 10μM HRP in 50Tris, 50Nacl, pH=7.5 buffer.
 +
#Take spectrum of the prepared 10μM HRP sample.
 +
#Add 2μL of Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub> to the prepared sample.
 +
#Take UV/vis spectrum of the sample.
 +
#Repeat steps 4-5 until complete oxidation of HRP.
 +
 +
==UV/vis==
  
  

Revision as of 11:15, 18 September 2013

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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.

Eleni Kalivas Tables For 091713 zemenk.png

UV/vis Spectra

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

NormalizedabsoxidatHRP09172013zem.png

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

ZoomedinnormalizedabsoxidationHRPzem.png

  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].

HRP Reduction

HRP Reduction Protocol

  1. Run UV-vis of a blank buffer sample.
  2. Prepare a 1mL sample of 10μM HRP in 50Tris, 50Nacl, pH=7.5 buffer.
  3. Take spectrum of the prepared 10μM HRP sample.
  4. Add 2μL of Na2S2O4 to the prepared sample.
  5. Take UV/vis spectrum of the sample.
  6. Repeat steps 4-5 until complete oxidation of HRP.

UV/vis