Date published: 2026-4-30

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3,3′,5,5′-Tetramethylbenzidine (CAS 54827-17-7)

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Alternate Names:
TMB substrate; BM blue; Sure Blue TMB
Application:
3,3′,5,5′-Tetramethylbenzidine is a sensitive reagent for the detection of hemoglobin
CAS Number:
54827-17-7
Molecular Weight:
240.34
Molecular Formula:
C16H20N2
For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.
* Refer to Certificate of Analysis for lot specific data.

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3,3′,5,5′-Tetramethylbenzidine (TMB) is a sensitive and specific reagent for the detection of blood, assay of hemoglobin, and assay of peroxidases. 3,3′,5,5′-Tetramethylbenzidine is a noncarcinogenic substitute (Ames test negative) for benzidine suitable as peroxidase substrate for use in ELISA procedures. The substrate produces a soluble end product that is pale blue in color and can be read spectrophotometrically at 370 or 620-650 nm. The TMB reaction may be stopped with 2 M H2SO4 (resulting in a yellow color), and read at 450 nm.


3,3′,5,5′-Tetramethylbenzidine (CAS 54827-17-7) References

  1. [Mechanisms of peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate].  |  Kireĭko, AV., et al. 2006. Bioorg Khim. 32: 80-6. PMID: 16523724
  2. Double wavelength measurement of 3,3',5,5'-tetramethylbenzidine (TMB) provides a three-fold enhancement of the ELISA measuring range.  |  Madersbacher, S. and Berger, P. 1991. J Immunol Methods. 138: 121-4. PMID: 1902244
  3. Investigation of 3,3',5,5'-tetramethylbenzidine as colorimetric substrate for a peroxidatic DNAzyme.  |  Li, B., et al. 2009. Anal Chim Acta. 651: 234-40. PMID: 19782817
  4. Highly sensitive and selective colorimetric detection of glutathione based on Ag [I] ion-3,3',5,5'-tetramethylbenzidine (TMB).  |  Ni, P., et al. 2015. Biosens Bioelectron. 63: 47-52. PMID: 25058938
  5. Selective extraction and release using (EDTA-Ni)-layered double hydroxide coupled with catalytic oxidation of 3,3',5,5'-tetramethylbenzidine for sensitive detection of copper ion.  |  Tang, S., et al. 2015. Anal Chim Acta. 885: 106-13. PMID: 26231895
  6. Selective sensing of submicromolar iron(III) with 3,3',5,5'-tetramethylbenzidine as a chromogenic probe.  |  Zhang, L. and Du, J. 2016. Spectrochim Acta A Mol Biomol Spectrosc. 158: 24-8. PMID: 26783724
  7. A simple and novel colorimetric assay for tyrosinase and inhibitor screening using 3,3',5,5'-tetramethylbenzidine as a chromogenic probe.  |  Lei, C., et al. 2017. Talanta. 175: 457-462. PMID: 28842016
  8. Colorimetric Sensor Arrays for Antioxidant Discrimination Based on the Inhibition of the Oxidation Reaction between 3,3',5,5'-Tetramethylbenzidine and Hydrogen Peroxides.  |  Li, X., et al. 2019. ACS Appl Mater Interfaces. 11: 9504-9509. PMID: 30735025
  9. A system composed of polyethylenimine-capped upconversion nanoparticles, copper(II), hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine for colorimetric and fluorometric determination of glyphosate.  |  Liu, Z., et al. 2019. Mikrochim Acta. 186: 835. PMID: 31758331
  10. Rationale of 3,3',5,5'-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis.  |  Zhang, X., et al. 2020. Anal Chem. 92: 12400-12406. PMID: 32794705
  11. 3,3',5,5'-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline values.  |  Palladino, P., et al. 2020. Anal Bioanal Chem. 412: 7861-7869. PMID: 32870352
  12. A colorimetric method for screening α-glucosidase inhibitors from flavonoids using 3,3',5,5'-tetramethylbenzidine as a chromogenic probe.  |  Liu, DM., et al. 2021. Colloids Surf B Biointerfaces. 197: 111400. PMID: 33113490
  13. Integration and Quantitative Visualization of 3,3',5,5'-Tetramethylbenzidine-Probed Enzyme-Linked Immunosorbent Assay-like Signals in a Photothermal Bar-Chart Microfluidic Chip for Multiplexed Immunosensing.  |  Fu, G., et al. 2021. Anal Chem. 93: 15105-15114. PMID: 34734693
  14. The use of 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate in microplate enzyme-linked immunosorbent assay.  |  Goka, AK. and Farthing, MJ. 1987. J Immunoassay. 8: 29-41. PMID: 3558827
  15. 3,3',5,5' - Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay.  |  Bos, ES., et al. 1981. J Immunoassay. 2: 187-204. PMID: 7047570

Ordering Information

Product NameCatalog #UNITPriceQtyFAVORITES

3,3′,5,5′-Tetramethylbenzidine, 500 mg

sc-208442
500 mg
$37.00
US: (Out of Stock: Availability April 2, 2026)

3,3′,5,5′-Tetramethylbenzidine, 1 g

sc-208442A
1 g
$53.00

3,3′,5,5′-Tetramethylbenzidine, 5 g

sc-208442B
5 g
$171.00

3,3′,5,5′-Tetramethylbenzidine, 25 g

sc-208442C
25 g
$515.00

3,3′,5,5′-Tetramethylbenzidine, 100 g

sc-208442D
100 g
$916.00

3,3′,5,5′-Tetramethylbenzidine, 250 g

sc-208442E
250 g
$2271.00