Western Blue® Stabilized Substrate for Alkaline Phosphatase From Promega

Western Blue® Stabilized Substrate for Alkaline Phosphatase is a stable, ready-to-use substrate for Western blots. The substrate is a mixture of BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium) and is stable in solution for up to one year. This is an improvement over traditional dried tablets of the individual components which have to be dissolved in water and used within an hour of dissolving. In the presence of alkaline phosphatase (generally conjugated to a secondary antibody), a purple substrate is deposited onto the membrane and easily visualized.

Our lab protocol for the entire Western blotting procedure takes approximately two days. On the first day, an SDS-PAGE gel is run. Proteins are transferred from the gel to PVDF or nitrocellulose membrane using laboratory protocol. In our lab, we use Biorad MiniProtean III gel system for SDS-PAGE and MiniTransBlot system for transfer of protein to membrane. We prefer PVDF membrane for our transfers. After transfer is complete, protein is blocked typically in a 5% milk solution in Tris-Buffered Saline pH7.5 (TBS: 50 mM Tris, 500 mM NaCl) for 1 hour followed by primary antibody in the same solution overnight with rocking at 4°C. Primary antibody dilution should be determined by the end user but we typically dilute our primary antibodies 1:1000 or 1:2000. After overnight incubation, we wash in TBS with 0.05% Tween-20. We do three washes at 10 minutes per wash with agitation. We then prepare secondary antibody solution in TBS/5% dried milk. Secondary antibody is typically diluted 1:5,000-1:10,000 but again should be determined by the user. Secondary antibody is left on the membrane for 1 hour at room temperature with gentle agitation (this can also be left on overnight but should be done at 4°C with agitation). The membrane is then washed in TBS with 0.05% Tween-20 3 times for 10 minutes each wash. We then wash a 4th time in deionized water and pour the water off. We then add 5-10 mls of substrate to the blot (this can be determined based upon the surface area of the membrane) and look for purple color formation. Color formation can take place in seconds to minutes depending on how much antibody is present on the membrane (which is dependent on the protein you are detecting). The reaction is stopped by simply washing away the substrate in water multiple times.

Background can be a problem particularly if the substrate is left on for a long time (for example 15-30 minutes) in order to be able to detect the protein of interest. Ideally, you would like exposure times of 1-3 minutes. This is dependent on several factors including how much starting protein is loaded onto the gel, how good your primary antibody is, and how long you wash the membrane. The dilutions and washes are recommended starting points but can be increased to remove background. Western blots for over-expressed proteins which contain a tag are ideally suited for this application. However, we have used this procedure to detect native proteins as well.