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

From OpenWetWare
Jump to navigationJump to search
Line 10: Line 10:
<br>
<br>
==General Protocol==
==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.
#Run UV-vis of a blank buffer sample.
#*Note:When preparing the sample add all components 
#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.  
(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.
#Take spectrum of the prepared 10μM HRP sample.
*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 2μL of K<sub>3</sub>[Fe(CN)<sub>6</sub>] to the prepared sample.
*solutions HRP that are 1mL 10μM when have 17.25μM starting.
#Take UV/vis spectrum of the sample.
#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 K<sub>3</sub>[Fe(CN)<sub>6</sub>] to the sample.




Revision as of 10:16, 17 September 2013

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Moira_M._Esson/Notebook/CHEM-571/2013/09/17&oldid=728943"