5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt, 500 mg
Molar mass (M) 433,63 g/mol
Melting point (mp) 198 °C
Storage temp. -20 °C
Transport temp. ambient temp.
CAS No. [6578-06-9]
Highly soluble in DMF (20 mg/ml), poorly soluble in water.
Protect from light and humidity.
excl. VAT. | 500 mg per Pack Qty.
Art. No. 6368.1
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5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt ≥99 %, BioScience Grade
In combination with NBT ideal for colorimetric detection of alkaline phosphatase activity, in immunohistochemical procedures.
By enzymatic dephosphorylation BCIP is converted to indoxyl which subsequently tautomerizes to the ketone. In basic pH values, this ketone dimerizes and, in doing so, releases H+ ions. The dye precipitates in the direct vicinity of the phosphatase, hence staining this site in a weak blue colour. Enhencement and darkening of this colour is achieved by adding NBT (Art. No. 4421).
|Application||Western, in situ, Histochemistry|
|Signal product||Precipitate (Blot)|
|Use||Western, in situ, histochimie|
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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.
|Appearance||white to almost white powder|
|Assay (titr.)||≥99 %|
|Water (KF)||≤1 %|
|Appearance of solution (1% in DMF)||farblos bis leicht gelblich, klar|
|Phosphate (PO4)||≤0,05 %|