Chemical Name: 3,3'-,5,5'-Tetramethylbenzidine
Appearance: White or pale grayish-brown crystalline powder
Purity: ≥99.0 % (HPLC)
MW: 240.34, C16H20N2
Storage Condition: ambient temperature
Shipping Condition: ambient temperature
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).
Assay Protocol for ELISA
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.
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