The proteins were separated by SDS-PAGE and then electrotransferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After incubation in blocking solution (5% skim milk) for 2 h, the membrane was incubated with a primary antibody overnight at 4°C. Subsequently, alkaline phosphatase-conjugated secondary antibody (Roche, Switzerland) was added and incubated for 2 h at room temperature. The membrane was rinsed and then incubated with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate (Sangon, China) until the blot was visualized.
5 bromo 4 chloro 3 indolyl phosphate bcip nitroblue tetrazolium nbt substrate
Manufactured by Sangon
Sourced in China
5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate is a chromogenic substrate used for the detection and visualization of alkaline phosphatase activity in various biological applications.
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2 protocols using 5 bromo 4 chloro 3 indolyl phosphate bcip nitroblue tetrazolium nbt substrate
1
Western Blot Protein Detection Protocol
2
Western Blot Analysis of Protein Expression
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The tissue was lysed in RIPA buffer (Beyotime, China), containing 1 mm PMSF for 30 min at 4 °C, and centrifuged at 12,000× g for 15 min at 4 °C. The tissue lysate was mixed with 5× SDS-PAGE buffer (Beyotime) and boiled for 10 min. Then, 30 μg of each sample was subjected to 10% SDS-PAGE and Western blotting, as previously reported [24 (link)]. The primary antibody diluted 1:1000 and secondary antibody diluted 1:2000 in 0.5% BSA-TBST. Following a further four 10 min washes in TBST, the membranes were detected with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate (Sangon Biotech, Shanghai, China). Each experiment was performed in triplicate.
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