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

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(HRP Reduction)
Current revision (14:18, 18 September 2013) (view source)
(UV/vis)
 
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==UV/vis==
==UV/vis==
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''' Normalized Absorbance spectrum of the reduction of HRP with sodium Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub>'''
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[[Image:NormalizedreductionHRPzem09172013.png|750px]]
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*Only 10uL were added because there were noted discrepancies in the group data.

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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.

Image: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]

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].

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

Normalized Absorbance spectrum of the reduction of HRP with sodium Na2S2O4

  • Only 10uL were added because there were noted discrepancies in the group data.




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