Chemical Name: 3,3'-,5,5'-Tetramethylbenzidine
CAS: 54827-17-7
Appearance: White or pale grayish-brown crystalline powder
Purity: ≥99.0 % (HPLC)
MW: 240.34, C16H20N2
Storage Condition: ambient temperature
Shipping Condition: ambient temperature
Product Description
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’- 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).
Oxidation Reaction
Assay Protocol for ELISA
<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)
<Procedure>
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.
References
1. V. R. Holland, et al., A Safer Substitute for Benzidine in the Detection of Blood. Tetrahedron. 1974;30:3299-3302.
2. W. Levin, et al., Preparation of Partially Purified, Lipid-depleted Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphatecytochrome c Reductase from Rat Liver Microsomes. J Biol Chem. 1974;249:1747-1754.
3. P. E. Thomas, et al., An Improved Staining Procedure for the Detection of the Peroxidase Activity of Cytochrome p-450 on Sodium Dodecyl Sulfate Polyacrylamide Gels. Anal Biochem. 1976;75:168-176.
4. H. H. Liem, et al., Quantitative Determination of Hemoglobin and Cytochemical Staining for Peroxidase Using 3, 3’, 5, 5’-Tetramethylbenzidine dihydrochloride, a Safe Substitute for Benzidine. Anal Biochem. 1979;98:388-393.
5. R. C. Lijana, et al., Tetramethylbenzidine- A substitute for Benzidine in Hemoglobin Analysis. J Lab Clin Med. 1979;94:266-276.
6. R. M. Jaffe, et al., A New Occult Blood Test Not Subject to False-Negative Results from Reducing Substances. J Lab Clin Med. 1979;93:879-886.
7. K. Suzuki, et al., Assay Method for Myeloperoxidase in Human Polymorphonuclear Leukocytes. Anal Biochem. 1983;132:345-352.
8. F. H. Pujol, et al., A Double Sandwich Monoclonal Enzyme Immunoassay for Detection of Hepatitis B Surface Antigen. J Immunoassay. 1993;14:21-31.
9. F. B. Serrat, Colorimetric Method for Determiantion of Chlorine with 3, 3’, 5, 5’-Tetramethylbenzidine. Talanta. 1994;41:2091-2094.