Steps in a Western blot:
- Gel electrophoresis - The proteins are separated according to size on a gel using SDS-PAGE. The gel has several lanes, and each lane contains one or more protein bands.
- Nitrocellulose transfer - The proteins in the gel are then transferred onto a membrane made of nitrocellulose or PVDF[?], by pressure or by applying a current. This is the actual blotting process and is necessary to expose the proteins. The membrane is "sticky" and binds proteins non-specifically (i.e., binds all proteins equally well).
- Blocking - The membrane is then blocked, suppressing non-specific protein interactions. This is done by a solution of BSA[?] or dry milk[?].
- The first antibody is applied. This antibody recognizes only the protein of interest, and will not bind any of the other proteins on the membrane. It is obtained by immunizing an animal with the protein of interest (i.e., injecting the protein into the animal's blood) and collecting the antibodies the animal produces against that protein. It is usually done with rabbits or goats.
- The second antibody is applied. It recognizes the first antibody, usually by its "producer", for example, α-rabbit (anti-rabbit) antibody might be used if the first antibody was produced by rabbits. The second antibody is usually produced in a different animal (i.e. goat anti-rabbit antibody). This antibody is linked to a chemical signal that can visually identify where on the membrane it has bound.
- The second antibody is then chemically stained. Although it is possible to have the chemical signal linked directly to the first antibody, it is better to separate the two functions (recognition and signalling) so that the two antibodies can be produced more easily.
Since the first antibody only recognizes the protein of interest, and the second antibody only recognizes the first antibody, if there is stain present on the membrane then the protein of interest must also be present on the membrane. Thus, the protein bands on the membrane that are stained contain the protein that was to be detected, the other bands do not. Size approximations can be done by comparing the staind bands to that of a pre-stained protein marker.
Usually, the gel is not completely devoid of proteins after blotting. Protein staining solution will show all protein bands on the gel. The stained gel can then be compared with the stained membrane to identify which bands contain the wanted protein and which do not.
Picture of a Western blot