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Line Structure
3D Ball and Stick
style="background: #F8EABA; text-align: center;" colspan="2" | Identifiers
CAS number 54827-17-7
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style="background: #F8EABA; text-align: center;" colspan="2" | Properties
Molecular formula C16H20N2
Molar mass 240.3482 g/mol
Melting point

168-171 °C

style="background: #F8EABA; text-align: center;" colspan="2" | Hazards
MSDS External MSDS
Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)
Infobox references

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)[1]. TMB is a white crystal powder which 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 sulphuric acid turns the product a yellow colour which may be read at 620 nm.

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,[2][3][4][5] and did not cause tumours in 24 rats[2] (but that experiment was uncontrolled). On that evidence, it has been used as a replacement for carcinogenic compounds such as benzidine[6] and o-phenylenediamine.[7]


  1. Sigma Aldrich Catalog Entry for 3,3′,5,5′-Tetramethylbenzidine
  2. 2.0 2.1 Holland VR, Saunders BC, Rose FL, Walpole AL (1974). "A safer substitute for benzidine in the detection of blood". Tetrahedron. 30: 3299. doi:10.1016/S0040-4020(01)97504-0. 
  3. Ashby J, Paton D, Lefevre PA, Styles JA, Rose FL (1982). "Evaluation of two suggested methods of deactivating organic carcinogens by molecular modification". Carcinogenesis. 3 (11): 1277–1282. doi:10.1093/carcin/3.11.1277. PMID 6758975. 
  4. Chung K-T, Chen S-C, Wong TY, Li YS, Wei CI, Chou MW (2000). "Mutagenicity studies of benzidine and its analogs: Structure-activity relationships". Toxicol Sci. 56 (2): 351–356. doi:10.1093/toxsci/56.2.351. PMID 10910993. 
  5. Chung K-T, Chen S-C, Claxton LD (2006). "Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes". Mutation Research/Reviews in Mutation Research. 612 (1): 58–76. doi:10.1016/j.mrrev.2005.08.001. PMID 16198141. 
  6. Yang J, Wang H, Zhang H (2008). "One-pot synthesis of silver nanoplates and charge-transfer complex nanofibers". J Phys Chem C. 112 (34): 13065–13069. doi:10.1021/jp802604d. 
  7. Deshpande SS (1996). Enzyme Immunoassays: From Concept to Product Development. New York: Chapman & Hall. p. 169. ISBN 978-0412056017.