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Hi,

Why does my elisa plate wells gives a brown color after adding the stop solution. I am giving a 4*5 minutes wash after each step. TMB gives it a proper blue color but when I add the stop solution (0.16M H2SO4), the color turns to brown. I am using all the reagents new. Any help will be appreciated.

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The full oxidation of TMB ( 3,5,3’,5’-tetramethylbenzidine) is 2-electron process. The first step (one electron oxidation) gives the blue product, which is then further oxidized to a yellow product (2nd electron) that is stable in acid. At high concentrations this might appear brown.

You might also not be fully quenching, leading to a mixture of blue and yellow products (should be more greenish though). In this case add more stop solution.

TMB chemistry:

Josephy, P. David, Thomas Eling, and Ronald P. Mason. "The horseradish peroxidase-catalyzed oxidation of 3, 5, 3', 5'-tetramethylbenzidine. Free radical and charge-transfer complex intermediates." Journal of Biological Chemistry 257.7 (1982): 3669-3675.

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