3,3’,5,5’-Tetramethylbenzidine
Encyclopedia
3,3’,5,5’-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry
as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA
). TMB is a white crystal powder that forms a pale blue-green liquid in solution with ethyl acetate.
In solution, TMB forms a blue product when allowed to react with peroxidase
enzymes such as horseradish peroxidase
. The resulting colour change can be read on a spectrophotometer at a wavelength of 370 or 655 nm. The reaction can be halted by addition of acid
or another stop reagent. Using sulfuric acid
turns TMB yellow. The colour may be read at 450 nm. TMB is also degraded by sunlight and by fluorescent lights.
and o-phenylenediamine
.
Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...
as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
). TMB is a white crystal powder that forms a pale blue-green liquid in solution with ethyl acetate.
In solution, TMB forms a blue product when allowed to react with peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
enzymes such as horseradish peroxidase
Horseradish peroxidase
The enzyme horseradish peroxidase , found in horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.-Applications:...
. The resulting colour change can be read on a spectrophotometer at a wavelength of 370 or 655 nm. The reaction can be halted by addition of acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...
or another stop reagent. Using sulfuric acid
Sulfuric acid
Sulfuric acid is a strong mineral acid with the molecular formula . Its historical name is oil of vitriol. Pure sulfuric acid is a highly corrosive, colorless, viscous liquid. The salts of sulfuric acid are called sulfates...
turns TMB yellow. The colour may be read at 450 nm. TMB is also degraded by sunlight and by fluorescent lights.
Material Safety
TMB should be kept out of direct sunlight as it is photosensitive. It is not known if TMB is carcinogenic and the evidence is contradictory: TMB is not mutagenic by the Ames test, and did not cause tumours in 24 rats (but that experiment was uncontrolled). On that evidence, it has been used as a replacement for carcinogenic compounds such as benzidineBenzidine
Benzidine, the trivial name for 4,4'-diaminobiphenyl, is the solid organic compound with the formula 2. This aromatic amine is a component of a test for cyanide and also in the production of dyes...
and o-phenylenediamine
O-Phenylenediamine
o-Phenylenediamine is a organic compound with the formula C6H42. This aromatic diamine is an important precursor to many heterocyclic compounds...
.