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Acid stop solution for HRP/TMB reaction


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#1 LOW

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Posted 06 September 2009 - 12:29 AM

HRP speeds up an electron transfer reaction. TMB is the donor and gets oxidized, and H2O2 is reduced to O2 and H2O.
H2SO4 denatures the enzyme as proteins dont survive under an extreme pH.


If the acid is just to denature the HRP, then why the blue colour of oxidized TMB changes yellow?

#2 swanny

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Posted 06 September 2009 - 05:15 PM

HRP speeds up an electron transfer reaction. TMB is the donor and gets oxidized, and H2O2 is reduced to O2 and H2O.
H2SO4 denatures the enzyme as proteins dont survive under an extreme pH.


If the acid is just to denature the HRP, then why the blue colour of oxidized TMB changes yellow?

Going back to undergraduate days (a long time ago), I think it's because, like many indicator compounds, oxidised TMB is pH-sensitive. I must admit I have never looked into it since then, so I'm quite possibly wrong, just another piece of minutiae that has stuck!
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