Elisa Method
We have TMB substrate which reacts with HRP and this reaction gives a blue priduct. Then we add Stop solution (HCl +H2SO4) and a yellow product is produced.1) Why does the colour change (why the λ changes)? 2) Why does the Absorbance increase? 3) Which is the role of acidification?
Hello Maria,
When in bottle, the TMB (which stands for 3,3’,5,5’- tetramethylbenzidine) is present in reduced form and is colorless. However, in presence of HRP (horse reddish peroxidase) and H2O2, the TMB gets oxidized and turn to blue. How it happens? The HRP converts H2O2 to water, obtaining the two hydrogen atoms it needs for this from a “donor” molecule i.e., TMB. Thus, at the same time that hydrogen peroxide is being reduced, the TMB is oxidized. Due to continuous reaction, the concentration of oxidized TMB becomes high enough to cause it to precipitate from the solution. Stop solution is therefore added to stabilize the color development for accurate measurement. Now for your points 1, 2, and 3, I would recommend you to go through following publication for it is too much chemistry to explain:
http://edoc.hu-berlin.de/oa/degruyter/cclm.1989.27.10.791.pdf
Hello Maria,
When in bottle, the TMB (which stands for 3,3’,5,5’- tetramethylbenzidine) is present in reduced form and is colorless. However, in presence of HRP (horse reddish peroxidase) and H2O2, the TMB gets oxidized and turn to blue. How it happens? The HRP converts H2O2 to water, obtaining the two hydrogen atoms it needs for this from a “donor” molecule i.e., TMB. Thus, at the same time that hydrogen peroxide is being reduced, the TMB is oxidized. Due to continuous reaction, the concentration of oxidized TMB becomes high enough to cause it to precipitate from the solution. Stop solution is therefore added to stabilize the color development for accurate measurement. Now for your points 1, 2, and 3, I would recommend you to go through following publication for it is too much chemistry to explain:
http://edoc.hu-berlin.de/oa/degruyter/cclm.1989.27.10.791.pdf
Is there any chance TMB may get oxidised by atmospheric oxygen even when H2O2 in the solution is exhausted?
2. Is H2SO4 attacking TMB or HRP to stop the reaction?
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