3,3'-Diaminobenzidine tetrahydrochloride, 1 g, glass
Molar mass (M) 360,10 g/mol
Melting point (mp) >280 °C
Storage temp. +4 °C
CAS No. [7411-49-6]
Application: Stock solution: 1-2 % (10-20 mg/ml) in 50 mM Tris buffer, pH 7.3. Storage of stock solution in aliquots at -20 °C. Working concentration: 0.05-0.1 % (0.5-1 mg/ml) DAB in buffer (PBS, TBS, pH 7.0-7.6) containing 0.01 % hydrogen peroxide. Always use freshly prepared working solution. For oxidation high concentrations of fresh hydrogen peroxide are needed.
excl. VAT. | 1 g per Pack Qty.
Art. No. CN75.1
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3,3'-Diaminobenzidine tetrahydrochloride ≥98 %, p.a.
Popular substrate of peroxidases. DAB is used for biochemical and histological detection of endogenous peroxidases like catalase and cytochome oxidase. Additionally, it is the most common substrate for horseradish peroxidase (HRP) in histochemical and cytological assays. DAB is oxidised by HRP, forming a brown precipitate, which is unsoluble in aqueous and organic solvents. This precipitate can be detected in visible light and does not bleach during long-term storage.
|Application||Western, in situ, Histochemistry|
|Signal product||Precipitate (Blot)|
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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.
|Assay (HPLC)||≥98 %|
|Suitablility for determination of Selenium||complies|
|Appearance of solution||Reddish brown|