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

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(HRP Oxidation)
Current revision (13:15, 18 September 2013) (view source)
(HRP Reduction)
 
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#*Note: No more than 16uL should be added of the Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub> to the sample.
#*Note: No more than 16uL should be added of the Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub> to the sample.
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<br>
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==UV/vis==
==UV/vis==
Figure 1. Normalized Absorbance spectrum of the reduction of HRP with sodium Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub>
Figure 1. 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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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]

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

  • 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].

HRP Reduction

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 Na2S2O4 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 16uL should be added of the Na2S2O4 to the sample.


UV/vis

Figure 1. Normalized Absorbance spectrum of the reduction of HRP with sodium Na2S2O4

  • Note: Only 10uL were added because there were noted discrepencies in the group data. Reduction of HRP will be repeated next lab.



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