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

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(General Protocol)
(General Protocol)
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# Find endpoints (100%) of oxidation and reduction of HRP  
# Find endpoints (100%) of oxidation and reduction of HRP  
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==General Protocol==
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==HRP Oxidation==
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'''General Protocol''':
#Run UV-vis of a blank buffer sample.
#Run UV-vis of a blank buffer sample.
#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.  
#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: Allow all samples to sit for approximately 5 minutes before taking the spectrum   
#*Note: Allow all samples to sit for approximately 5 minutes before taking the spectrum   
#*Note: No more than 20uL should be added of the K<sub>3</sub>[Fe(CN)<sub>6</sub>] to the sample.
#*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


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.




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