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

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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  K<sub>3</sub>[Fe(CN)<sub>6</sub>]. K<sub>3</sub>[Fe(CN)<sub>6</sub>], which has a reduction potential of 424mV. HRP reduction will be done with Sodium Dithionite.  
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  K<sub>3</sub>[Fe(CN)<sub>6</sub>]. K<sub>3</sub>[Fe(CN)<sub>6</sub>], 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.
* 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 K<sub>3</sub>[Fe(CN)<sub>6</sub>] 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]'''
[[Image:NormalizedabsoxidatHRP09172013zem.png|750px]]
'''Figure 2: Enlarged Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]'''
[[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].
=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==
''' Normalized Absorbance spectrum of the reduction of HRP with sodium Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub>'''
[[Image:NormalizedreductionHRPzem09172013.png|750px]]
*Only 10uL were added because there were noted discrepancies in the group data.


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Revision as of 11:18, 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.

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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