User:Moira M. Esson/Notebook/CHEM-571/2013/09/17: Difference between revisions
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==Objectives== | ==Objectives== | ||
# Find endpoints (100%) of oxidation and reduction of HRP | |||
<br> | |||
==General Protocol== | |||
#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 | *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 09:57, 17 September 2013
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Objectives
General Protocol
(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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