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

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==Objectives==
==Objectives==
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* Find endpoints of HRP (sodium dithionite will reduce and potassium ferrocyanide will oxidize).All buffers are degassed (bubbled nitrogen through for 3 hours)---endpoints of oxidation and reduction of HRP. look for 100% oxidized and 100% reduced.
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# Find endpoints (100%) of oxidation and reduction of HRP  
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<br>
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==General Protocol==
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#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.
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#*Note:When preparing the sample add all components 
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(sodium dithionite will reduce and potassium ferrocyanide will oxidize. All buffers are degassed (bubbled nitrogen through for 3 hours)---endpoints of oxidation and reduction of HRP. look for 100% oxidized and 100% reduced.
*Add very carefully and very slowly and then just let it sit for about 5 minutes before take the sample. Need blank spectra(buffer) of the cuvette
*Add very carefully and very slowly and then just let it sit for about 5 minutes before take the sample. Need blank spectra(buffer) of the cuvette
*solutions HRP that are 1mL 10μM when have 17.25μM starting.  
*solutions HRP that are 1mL 10μM when have 17.25μM starting.  

Revision as of 12:57, 17 September 2013

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Objectives

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


General Protocol

  1. 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.
    • Note:When preparing the sample add all components

(sodium dithionite will reduce and potassium ferrocyanide will oxidize. All buffers are degassed (bubbled nitrogen through for 3 hours)---endpoints of oxidation and reduction of HRP. look for 100% oxidized and 100% reduced.

  • Add very carefully and very slowly and then just let it sit for about 5 minutes before take the sample. Need blank spectra(buffer) of the cuvette
  • solutions HRP that are 1mL 10μM when have 17.25μM starting.



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