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3,3',5,5'-Tetramethylbenzidine dihydrochloride, 1 g

≥99 %, for biochemistry
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TMB · 2 HCl, TMB dihydrochloride
Empirical formula C16H20N2 · 2 HCl
Molar mass (M) 313,27 g/mol
Melting point (mp) 300 °C
Storage temp. -20 °C
WGK 1
CAS No. [207738-08-7]
EG-Nr. 264-769-6

Water-soluble peroxidase substrate
Stock solution: 0.1 mg/ml in 50 % acetic acid or phosphate citrate buffer (50 mM NaH2PO4, 25 mM citric acid, pH 5.0). For solubilisation, heat to 45 °C and sonicate. Colourless.

Storage of stock sol.: At +4 °C if tightly sealed. Oxidation can be seen by colour-shift to a faint blue.

Working solution: Immediately prior to use add 2 µl / 10 ml of 30 % hydrogen peroxide.

(Liem et al. (1979) Anal Biochem, 98:388-93).

Tetramethylbenzidine dihydrochloride is used as highly sensitive substrate for detection of horseradish peroxidase labelled probes, particularly in solution-based assays like ELISA techniques. It produces a soluble, blue-coloured end product, which can be read spectrophotometrically. The dihydrochloride form is well water-soluble, making it a versatile tool in laborartory science.
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Art. No. 4107.2

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Product details



3,3',5,5'-Tetramethylbenzidine dihydrochloride ≥99 %, for biochemistry

Tetramethylbenzidine dihydrochloride is used as highly sensitive substrate for detection of horseradish peroxidase labelled probes, particularly in solution-based assays like ELISA techniques. It produces a soluble, blue-coloured end product, which can be read spectrophotometrically. The dihydrochloride form is well water-soluble, making it a versatile tool in laborartory science.



Technical Information
Enzyme Horseradish peroxidase 
Detection Colour (blue) 
Signal product soluble (ELISA) 
3,3',5,5'-Tetramethylbenzidine dihydrochloride
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4107.1 500 mg glass €42.50
4107.2 1 g glass €68.70
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Downloads / MSDS


General information

Assay protocol for use of BCIP/NBT in immunoblot procedures:

Stock solutions (All solutions are stable for at least 1 year at 4 °C):
0.5 g NBT in 10 ml 70 % dimethylformamide;
0.5 g BCIP (p-toluidine salt) in 10 ml 100 % dimethylformamide.
Incubation buffer for alkaline phosphatase:
100 mM NaCl, 5 mM MgCl2,100 mM Tris (pH 9.5).
Fresh substrate solution: 66 μl NBT stock solution + 10 ml incubation buffer, mix well, add 33 μl BCIP stock solution. Use within 1 hour.
Blot development: Approx. 10 ml substrate solution per 15 x 15 cm2 membrane surface. Develop at room temperature until bands become visible (approx. 30 mins).
Reaction stop: Rinse with PBS/20 mM EDTA.


Detection of peroxidase activity (Immunoassay):

Dissolve 1 mg TMB in 0.1 ml dimethylsulfoxide (Art. No. 4720); add 9.9 ml of a 0.1 M sodium acetate solution (pH 6.0) (Powder: Art. No. 6779), filter and add H2O2 (Art. No. 8070) (final concentration 0.01 %).

Always prepare freshly!

Incubation 10-30 mins at room temperature (approx. 50 μl per microtitre well; finally add 50 μl 1 M H2SO4, Art. No. X873, per well).
Photometric quantitation at 450 nm.

Reference: Bos, E.S. et al., (1981) J. Immunoassay 2, (3/4), 187.


Certificates of Analysis

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Guarantee analysis

Assay (titr.)≥99 %
Solution (1 % in methanol)colourless to yellow-green
Water (KF)≤5 %
Sulphated ash≤0,1 %

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