Final answer:
NBT/BCIP is used as a chromogenic substrate in western blotting to detect proteins. It reacts with enzymes conjugated to secondary antibodies, producing colored precipitates at the protein bands.
Step-by-step explanation:
In western blotting, NBT/BCIP (Nitro Blue Tetrazolium/5-Bromo-4-Chloro-3'-Indolyphosphate) is a chromogenic substrate used to detect proteins. After proteins are separated by gel electrophoresis and transferred onto a nitrocellulose or PVDF membrane, the membrane is incubated with primary antibodies that specifically bind to the target protein. Then, secondary antibodies conjugated with enzymes like horseradish peroxidase (HRP) or alkaline phosphatase (AP) are applied. When NBT/BCIP is added, the enzymes catalyze a reaction that produces a purple or blue precipitate at the site of protein bands, indicating the presence of the target protein.
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