p-Nitrotetrazolium blue chloride, 100 mg
Molar mass (M) 817,65 g/mol
Melting point (mp) 189 °C
Storage temp. +4 °C
CAS No. [298-83-9]
excl. VAT. | 100 mg per Pack Qty.
Art. No. 4421.1
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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.
|Application||Western, in situ, Histochemistry|
|Signal product||Precipitate (Blot)|
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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.
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 (NT)||≥98 %|
|Water (KF)||≤5,0 %|