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

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(General Protocol)
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==General Protocol==
==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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#Run UV-vis of a blank buffer sample.
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#*Note:When preparing the sample add all components 
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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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(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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#Take spectrum of the prepared 10μM HRP sample.
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*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
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#Add 2μL of K<sub>3</sub>[Fe(CN)<sub>6</sub>] to the prepared sample.
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*solutions HRP that are 1mL 10μM when have 17.25μM starting.
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#Take UV/vis spectrum of the sample.
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#Repeat steps 4-5 until complete oxidation of HRP.
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#*Note: When preparing the sample add all components '''very'' slowly
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#*Note: Allow all samples to sit for approximately 5 minutes before taking the spectrum 
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#*Note: No more than 20uL should be added of the K<sub>3</sub>[Fe(CN)<sub>6</sub>] to the sample.
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Revision as of 13:16, 17 September 2013

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Objectives

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


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.



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