SIGMAFAST™ BCIP®/NBT B5655 Tablets From Sigma-Aldrich

The technique of Western blotting is a laboratory staple of protein expression analysis. After loading protein samples onto an appropriate gel and separation by electrophoresis, the gel is then blotted onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane, for subsequent probing with appropriate antibodies. In addition, porous membranes made of cellulose acetate can be used to trap aggregates present in protein samples for antibody detection by using filter trap or dot blotting techniques. Usually, after probing the membrane with a primary antibody specific to the protein of interest, a secondary antibody coupled to the enzyme horseradish peroxidase (HRP) serves to detect the protein. This enzyme catalyzes the oxidation of the enhanced luminol reagent added in the presence of an oxidizing reagent, resulting in the emission of light. This localized light emission creates an image on X-ray film at the location of the protein on the blot, which can be visualized following exposure in a dark room and development in a film processor.

The BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) tablets represent a colorimetric alternative to the HRP-mediated, light-based detection (i.e. chemiluminescence). The tablets are a precipitating substrate for the detection of alkaline phosphatase activity. These tablets are reported as requiring no additional buffers or preparation steps. To generate an active substrate solution, simply reconstitute the tablets. For reconstitution, one tablet dissolved in 10 mls deionized water yields a ready-to-use buffered solution containing BCIP/NBT at pH 9.5.

The blot is processed as usual, with incubations in blocking buffer and the primary antibody being interspersed with appropriate washings with TBS/Tween buffer. The secondary antibody used is coupled to an alkaline phosphate instead of HRP, and can be applied in dilutions as low as 1:30,000. The blot is then washed (5-6 washes of 15 mins each), before incubating in the BCIP/NBT solution for development. A dark purplish band indicates the presence of the detected protein; the reaction is stopped by replacing the BCIP/NBT solution with deionized water.

The BCIP/NBT tablets can essentially be used wherever the HRP system was used previously, and therefore has obvious applications in immunochemistry, immunoblotting, and dot blotting. The system has many advantages over other conventional methods of immunoblot protein detection. For one, I have found the reconstituted buffer to be very sensitive to the alkaline phosphatase reaction, allowing for very high dilutions and thus, conserving use of secondary antibody and. The color development usually takes place in about 10-15 minutes, at which time the reaction is stopped. Another obvious advantage over the HRP system is that the use of X-ray film and film processors is obviated, since protein detection is developed on the blot itself. It is thus a much cheaper alternative, and loss of signal due to less than optimal transfer to film is prevented. However, it does suffer from some drawbacks, the most significant one in my estimation is that it does not allow for stripping and reprobing with alternative antibodies. It may also be best for detection of protein expression, rather than quantification or comparison of expression levels.