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

≥98 %, p.a.
Pack Qty.
Empirical formula C16H20N2
Molar mass (M) 240,35 g/mol
Melting point (mp) 170 °C
Storage temp. +4 °C
CAS No. 54827-17-7
EG-Nr. 259-364-6

Substrate for peroxidase
Always prepare a fresh working solution!

Used as a highly sensitive substrate for peroxidases, particularly in ELISA procedures. Can replace carcinogenic benzidine.
Product details

€62.25/Pack Qty. 

excl. VAT. | 1 g per Pack Qty.

Art. No. 6350.1

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Product details

3,3',5,5'-Tetramethylbenzidine ≥98 %, p.a.

Used as a highly sensitive substrate for peroxidases, particularly in ELISA procedures. Can replace carcinogenic benzidine.

Dissolves easily in acetone, chloroform and ethanol.

Directions for use

Working solution: 1 mg TMB in 0.1 ml dimethyl sulfoxide, add 9.9 ml 0.1 M sodium acetate (pH 6). Filter and add H2O2 ad 0.01 %. Always prepare freshly!
Application: approx. 50 µl per 96well, incubate for 10-30 mins. at room temperature. Add 50 µl 1 M H2SO4 and quantitate photometrically at 450 nm (acc. to: Bos et. al. (1981) J. Immunoassay 2:187).
Protect from light.

Technical Information
Enzyme Horseradish Peroxidase 
Detection Colour (blue) 
Signal product soluble (ELISA) 
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Art. No. Pack Qty. Pack. Price Quantity
6350.1 1 g glass


6350.2 5 g plastic


6350.3 25 g plastic


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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

You can search for and download your certificate of analysis for the selected product here. Please provide your batch number.
The following analysis certificates have been found:

Type analysis

Appearancebeige cryst. powder
Assay (GC)≥99.0 %
Sulphated ash≤0,2 %
Loss on drying (3 h, 60 °C, vacuum)≤1,0 %
Melting range166-171 °C
Can replace carcinogenic benzidine.