User:Megan L. Channell/Notebook/Horseradish/2013/09/17

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Oxidation and Reduction HRP

Objective

  • Find the endpoints of 100% oxidized and 100% reduced HRP.
  • Oxidation uses potassium ferricyanide
  • Reduction uses sodium dithionite

Protocol

Oxidation

  1. Take a blank UV-Vis spectrum of Tris Buffer
  2. Make a 1 mL HRP solution in cuvette with concnetration of 10μM (HRP=.5797mL and Buffer=.4203mL)
    1. Wait 5 mins before running on UV-Vis
    2. Add all regents slowly without adding bubbles into the cuvette
    3. Take spectrum
  3. Add 2μL of K3[Fe(CN)6]
    1. Wait 5 mins
    2. Take spectrum
  4. Reapeat for a total of 8x so the final volume of K3[Fe(CN)6] in cuvette is 16μL

Reduction

  1. Follow same procedure above except instead of using K3[Fe(CN)6], use sodium dithionite

Data

UV-Vis from 800 to 200 nm of HRP Reduction Image:9 17 2013 Reduction cmj HRP.png

UV-Vis from 800 to 200 nm of HRP Oxidation Image:9 17 2013 oxidation cmj HRP.png

Image:9 17 2013 cmj concentartions.png


Note: HRP stock is 17.2 mM The oxidation with Potassium Ferricyanide was for the most part oxidized. The little shift occurred after the first adding of Potassium Ferricyanide. This was seen across the entire group data. Dr. Harting's was able to determine the reduction through a more concentrated starting amount of sodium dithionite. His procedure for this is here and this will carry us into 9/18/2013.


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