User:Moira M. Esson/Notebook/CHEM-571/2013/09/17

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Figure 1. Normalized Absorption Spectra of the Oxidation of HRP with K<sub>3</sub>[Fe(CN)<sub>6</sub>]
Figure 1. Normalized Absorption Spectra of the Oxidation of HRP with K<sub>3</sub>[Fe(CN)<sub>6</sub>]
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[[Image:OxidationHRP09172013zem.png]]
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[[Image:NormalizedabsoxidatHRP09172013zem.png|500]]
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Figure 2. Enlarged Absorption Spectra of the Oxidation of HRP with K<sub>3</sub>[Fe(CN)<sub>6</sub>]
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[[Image:ZoomedinnormalizedabsoxidationHRPzem.png|500]]
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*Initial drop in absorbance occurred after the addition of 2uL K<sub>3</sub>[Fe(CN)<sub>6</sub>].
*Initial drop in absorbance occurred after the addition of 2uL K<sub>3</sub>[Fe(CN)<sub>6</sub>].
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*100% oxidation occurred (absorbance shift) after the addition of 6uL K<sub>3</sub>[Fe(CN)<sub>6</sub>].
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*The addition of 2uL of K<sub>3</sub>[Fe(CN)<sub>6</sub>] caused the complete oxidation of HRP. This indicates that HRP was mostly oxidized prior to additon of K<sub>3</sub>[Fe(CN)<sub>6</sub>].  

Revision as of 12:23, 18 September 2013

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Objectives

  1. Find endpoints (100%) of oxidation and reduction of HRP


HRP Oxidation

General 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. This buffer should be degassed) directly in a clean glass cuvette.
  3. Take spectrum of the prepared 10μM HRP sample.
  4. Add 2μL of K3[Fe(CN)6] to the prepared sample.
  5. Take UV/vis spectrum of the sample.
  6. Repeat steps 4-5 until complete oxidation of HRP.
    • Note: When preparing the sample add all components 'very slowly
    • Note: Allow all samples to sit for approximately 5 minutes before taking the spectrum
    • Note: No more than 20uL should be added of the K3[Fe(CN)6] to the sample.


Table 1: Sample preparation for HRP

Dilution 'M1'[μM] 'V1' [mL] 'M2'[μM] 'V2' [mL]
117.25.580101
  • Added .419mL of buffer to reach a volume of 1mL.


UV/vis

Figure 1. Normalized Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]
500
Figure 2. Enlarged Absorption Spectra of the Oxidation of HRP with K3[Fe(CN)6]
500

  • Initial drop in absorbance occurred 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].



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