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p-Nitrotetrazolium blue chloride, 100 mg

≥98 %, p.a.
Pack Qty.
Pack.
Nitro-BT, NBT, Nitroblue tetrazolium chloride
Empirical formula C40H30Cl2N10O6
Molar mass (M) 817,65 g/mol
Melting point (mp) 189 °C
Storage temp. +4 °C
WGK 1
CAS No. [298-83-9]
EG-Nr. 206-067-4

Application: Stock solution: 0.5 g NBT in 10 ml 70 % dimethylformamide. Protect from light and humidity.

Used in combination with BCIP-toluidine salt (X-phosphate) for detection of alkaline phosphatase activity by producing an indigo dye precipitate. Colour enhancer for staining with alkalic phosphatase. During dimerisation of ketonized X-Phos, H+ ions are set free which reduce NBT to purple-coloured diformazan. Both dyes precipitate in the direct vicinity of the phosphatase, hence staining this site in a dark violet. Store at +4 °C. Please also see assay protocol for use of BCIP/NBT in immunoblot procedures.
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€18.85/Pack Qty. 

excl. VAT. | 100 mg per Pack Qty.

Art. No. 4421.1

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Product details



p-Nitrotetrazolium blue chloride ≥98 %, p.a.

Used in combination with BCIP-toluidine salt (X-phosphate) for detection of alkaline phosphatase activity by producing an indigo dye precipitate. Colour enhancer for staining with alkalic phosphatase. During dimerisation of ketonized X-Phos, H+ ions are set free which reduce NBT to purple-coloured diformazan. Both dyes precipitate in the direct vicinity of the phosphatase, hence staining this site in a dark violet. Store at +4 °C. Please also see assay protocol for use of BCIP/NBT in immunoblot procedures.



Technical Information
Application Western, in situ, Histochemistry 
Enzyme Alkaline phosphatase 
Detection Colour (brown-violet) 
Signal product Precipitate (Blot) 
p-Nitrotetrazolium blue chloride
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Order No. Pack Qty. Pack. Price Quantity
4421.1 100 mg glass €18.85
4421.2 500 mg glass €48.30
4421.3 1 g glass €88.70
4421.4 5 g glass €321.45
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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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The following analysis certificates have been found:

Type analysis

Assay (HPLC)≥98 %
Water (KF)≤5.0 %

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