05 Cell Staining
10 Redox Dyes



Coloring Substrate for Peroxidase

  • Product code
    T022  TMBZ
  • CAS No.
  • Chemical name
  • MW
Unit size Price Item Code
1 g $80.00 T022-10
5 g $285.00 T022-12


TMBZ is a chromogenic reagent utilized for peroxidase detection. It has been developed as an alternative to benzidine, which is a carcinogenic chemical. Because of the ortho methyl groups on its benzene ring, TMBZ is not metabolized into highly carcinogenic o-hydroxybenzidines or o,o E dihydroxybenzidines. Therefore, TMBZ compounds are much less carcinogenic than benzidine. Although the TMBZ solution is colorless, it turns bluish-green (λmax: 655 nm) in the presence of hydrogen peroxide and peroxidase. The structure of this bluish-green complex is thought to be a radical form of two oxidized TMBZ molecules. TMBZ HCl is a hydrochloride form of TMBZ that is readily soluble in water (100 mg TMBZ HCl per 10 ml water).

Technical info

Substrate solution
Prepare 1 mL of 55mmol/L TMBZ in DMSO solution, then mix with 100 mL of 0.1 mol/L Sodium Acetate buffer (pH5)

1. Add Substrate solution to each well of 96 well plate contain POD labeled antibody, then incubate the plate at 30 degree C for 5 minutes.
2. Add 200 ul of 0.1 g/L H2O2, then incubate the plate at 30 degree C for 10 – 100 minutes.
3. Add 200 ul of 2 mol/L H2SO4 to stop the enzyme reaction. Read absorbance at 450 nm.


Open References

1) V. R. Holland, B. C. Saunders, F. L. Rose and A. L. Walpole, "A Safer Substitute for Benzidine in the Detection of Blood", Tetrahedron, 1974, 30, 3299. 
2) W. Levin, D. Ryan, S. West and A. Y. H Lu, "Preparation of Partially Purified, Lipid-depleted Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphate-cytochrome c Reductase from Rat Liver Microsomes", J. Biol. Chem., 1974, 249, 1747. 
3) P. E. Thomas, D. Ryan and W. Levin, "An Improved Staining Procedure for the Detection of the Peroxidase Activity of Cytochrome p-450 on Sodium Dodecyl Sulfate Polyacrylamide Gels", Anal. Biochem., 1976, 75, 168. 
4) H. H. Liem, F. Cardenas, M. Tavassoli, M. B. Poh-Fitzpatrick and U. Muller-Eberhard, "Quantitative Determination of Hemoglobin and Cytochemical Staining for Peroxidase Using 3,3',5,5'-Tetramethylbenzidine dihydrochloride, a Safe Substitute for Benzidine", Anal. Biochem., 1979, 98, 388. 
5) R. C. Lijana and M. C. Williams, "Tetramethylbenzidine- A substitute for Benzidine in Hemoglobin Analysis", J. Lab. Clin. Med., 1979, 94, 266. 
6) R. M. Jaffe and W. Zierdt, "A New Occult Blood Test Not Subject to False-Negative Results from Reducing Substances", J. Lab. Clin. Med., 1979, 93, 879. 
7) 吉野二男, "還元剤によっても偽陰性を示さない新しい潜血反応試薬", 臨床検査, 1980, 24, 140. 
8) K. Suzuki, H. Ota, S. Sasagawa, T. Sakatani and T. Fujikura, "Assay Method for Myeloperoxidase in Human Polymorphonuclear Leukocytes", Anal. Biochem., 1983, 132, 345. 
9) 鈴木和男, 笹川澄子, 坂谷達一郎, 太田洋美, 大西寿, 藤倉敏夫, "自動マルチウェルリーダーを用いた酵素活性の高感度レートアッセイ法 -多形核白血球ミエロパーオキシデースをモデルとして", 医学のあゆみ, 1984, 131, 163. 
10) F. H. Pujol, I. Rodriguez, M. Devesa, R. Rangel-Aldao and F. Liprandi, "A Double Sandwich Monoclonal Enzyme Immunoassay for Detection of Hepatitis B Surface Antigen", J. Immunol., 1993, 14, 21. 
11) F. B. Serrat, "Colorimetric Method for Determiantion of Chlorine with 3,3',5,5'-Tetramethylbenzidine", Talanta, 1994, 41, 2091.

Handling and storage condition

Appearance: White to pale grayish brown crystalline powder
Purity (HPLC): ≧ 99.0 %
Solubility in Methyl alcohol: To pass test (clear, colorless to slightly yellow)
Molar absorptivity: ≧ 24,000 (around 287 nm)
m.p.: 165 - 171oC
IR spectrum: Authentic
Handling and storage condition
Ambient temperature

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