BCIP/NBT Color Development Substrate
(5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium)

BCIP (5-bromo-4-chloro-3-indolyl-phosphate) is used in conjunction with NBT (nitro blue tetrazolium) for the colorimetric detection of alkaline phosphatase activity. Each vial of BCIP is supplied with a vial of NBT.

Preparation of Substrates to Detect Alkaline Phosphatase: For every 5ml of alkaline phosphatase buffer (100mM Tris-HCl [pH 9.0], 150mM NaCl, 1mM MgCl₂), add 33μl NBT and 16.5μl BCIP. Add the NBT first, mix, add the BCIP, and mix again. Use within 1 hour, and discard any unused solution.

Concentration: BCIP (50mg/ml) in 100% dimethylformamide; NBT (50mg/ml) in 70% dimethylformamide.