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.
- After incubating the Western blot in primary antibody, wash the nitrocellulose membrane four times for 5 minutes each, with sufficient TBS/TWEEN.
- Incubate the membrane for 1 hour in alkaline phosphatase-secondary antibody conjugate at an appropriate dilution. (1:30,000 for upgraded alkaline phosphatase conjugates) in TBS/TWEEN.
- Wash the membrane 4 times in TBS for 5 minutes each.
- Prepare SIGMAFAST™ BCIP/NBT Tablets (Product No. B5655) according to package directions. Liquid BCIP/NBT substrate may also be used (Product No. B1911).
- Incubate the membrane in the substrate mixture for 10-30 minutes until color development.
- Stop the reaction by washing the membrane in several changes of distilled water.
- Air dry the membrane and store in the dark in a plastic sleeve.
Note: The dry membrane may be stored at 2-8 °C between two sheets of blotting paper in a plastic sleeve.