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#TMB

2 public questions tagged with this topic.

Blotting Techniques

The enzyme Horseradish Peroxidase (HRP) with TMB produces:

Horseradish peroxidase is a heme-containing oxidoreductase widely conjugated to secondary antibodies for Western detection. With hydrogen peroxide as electron acceptor, HRP oxidizes chromogenic substrate 3,3',5,5'-tetramethylbenzidine. Oxidation yields a cation radical forming a blue charge-transfer complex with maximal absorbance at 652 nm soluble or as precipitate on membrane. Reaction is rapid, sensitive, and reversible upon acidification with sulfuric acid, converting blue product to yellow diamine measurable at 450 nm. Alternative substrates give distinct colors such as brown from diaminobenzidine and purple from BCIP/NBT with alkaline phosphatase.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Microscopy

In ELISA, what color is produced by HRP and TMB?

Horseradish peroxidase in presence of hydrogen peroxide oxidizes 3,3',5,5'-tetramethylbenzidine through single electron transfer steps. Initial oxidation yields a cation radical forming charge transfer complex that appears blue with absorption maxima near 650 nm. This intermediate is stable enough for kinetic reading without stopping. Addition of sulfuric acid halts reaction by denaturing enzyme and further oxidizes TMB to yellow diimine absorbing at 450 nm. Purple or brown precipitates belong to other systems like BCIP-NBT or DAB. Thus native peroxidase-TMB interaction generates distinctive blue, widely accepted for ELISA endpoint quantification.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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