Chemical Name: 3,3'-,5,5'-Tetramethylbenzidine, dihydrochloride, dihydrate
Appearance: white or pale yellow powder
Purity: >98.0% (Titration)
MW: 349.30,C16H22Cl2N2· 2H2O
Storage Condition: ambient temperature
Shipping Condition: ambient temperature
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).
1. Dissolve 6 mg of TMBZ with 1 ml DMSO to prepare 100X TMBZ solution.
2. Mix 5 μl of 30% hydrogen peroxide solution with 1 ml PBS to prepare 200X H2O2 solution.
3. Add 10 μl of 100X TMBZ solution and 5 μl of 200X H2O2 solution to 1 ml PBS to prepare staining solution.*
Add 100-200 μl of the staining solution to each well of a 96-well
microplate. Incubate the plate at room temperature or at 37ºC for 5
minutes to 1 hour.
5. Wash the sample with PBS several times to stop the staining reaction.
For the best results, modification of the final concentration of TMBZ
and hydrogen peroxide may be necessary. The staining solution is not
stable. Please prepare fresh solution prior to use.
1. V. R. Holland, et al., A Safer Substitute for Benzidine in the Detection of Blood. Tetrahedron. 1974;30:3299-3302.
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.
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
4. H. H. Liem, et al., Quantitative
Determination of Hemoglobin and Cytochemical Staining for Peroxidase
Using 3, 3 E 5, 5 ETetramethylbenzidine 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.
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.
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 E 5, 5 ETetramethylbenzidine. Talanta. 1994;41:2091-2094.