Immunoblotting, often known as western blotting, is a technique for separating proteins by molecular weight. Two gels are created in this technique: a stacking gel pH (6.8) and a separating gel pH (8.8). The samples are put onto an acidic stacking gel, which separates proteins poorly but allows them to form narrow, highly defined bands. Smaller proteins travel quicker than larger protein molecules on a separating or resolving gel used to separate proteins based on their size. When voltage is supplied to the protein before loading, it is denatured and has a negative charge that travels towards the positive electrode. After the protein separation, the gel is sliced and inserted in a sandwich of fibre pad and filter sheets with a membrane (PVDF or nitrocellulose) and placing it in the tank with the transfer buffer with overnight transfer of protein to the membrane. After the protein has been transferred to the membrane, it is blocked by blocking solution to prevent antibodies from attaching nonspecifically to the membrane. Washing is done three times at a 10-minute interval after blocking. After that, the primary antibody is added for four hours, and then washing is done three times at a five-minute interval. The membrane is then added with a secondary antibody. The membrane is then detected using the label antibody and an enzyme such as horseradish peroxidase (HRP), which is identified by the signal it creates matching to the target protein's position. This signal is recorded on film, which is typically developed in a dark environment and hence blots are developed.
The requirement to test more proteins in fewer samples at the same time has prompted continued research towards increasing the sensitivity and speed of blotting techniques. Nonspecific interactions create false positives, which are eliminated by double blotting. Specific protein-protein interactions can be detected via Far-Western blotting. Proteins that interact with certain DNA sequences are identified by southwestern blotting. Multistrip blotting improves throughput while reducing variability between blots. New methods are being developed to lower the amount of protein required to produce a signal and to improve Western blotting's quantitative capabilities.