5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) is an ideal insoluble substrate for use with alkaline phosphatase. This system produces a blue-purple product. The intense color can be observed visually, is very stable, and will not fade upon exposure to light.
The following Western blot incubation and washing steps are carried out at room temperature on an orbital shaker platform. Primary antibody is diluted in 1% normal serum from the secondary antibody host animal in TBS. Other non-interfering proteins (e.g., BSA, hemoglobin, ovalbumin) may be substituted. If using PVDF membrane, conditions may require optimization.
Note: The dry membrane may be stored at 2-8 °C between two sheets of blotting paper in a plastic sleeve.
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