Western blot
A
Western blot is a method in
molecular biology to detect a certain
protein in a sample by using
antibody specific to that protein. It also gives information about the size of that protein. Its name is a pun off the name
Southern blot, a similar technique developed earlier by Ed Southern.
Steps in a Western blot:
- Gel electrophoresis - The proteins of the sample are separated according to size on a gel using SDS-PAGE. The gel has several lanes so that several samples can be tested simultaneously, 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. (Without the blocking, the antibody to be applied in the next step would bind to the nitrocellulose.)
- 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 (usually a rabbit or goat) 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.
- The second antibody is applied. It binds to the first antibody, and is usually produced by a different animal. For example, goat anti-rabbit antibody might be used if the first antibody was produced by rabbits. This second antibody is linked to a chemical signal that can visually identify where on the membrane it has bound. Similar to the ELISA procedure, this chemical signal is often an enzyme which can produce fluorescence in its substrate.
- Free antibody is washed away, and a substance is added to the membrane so that the second antibody will become visible.
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.
Picture of a Western blot with 5 vertical lanes
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.
In principle, one could bind the chemical signal directly to the first antibody, but production of the antibodies is easier if the two functions recognition and signalling are separated.
The HIV test known as "Western Blot" uses a variant of the technique, where the goal is to detect the presence of antibody in a sample. Known HIV infected cells are opened and their proteins separated and blotted on a membrane as above. Then the serum to be tested is applied. Free antibody is washed away, and a secondary antibody is added that binds to human antibody and is linked to an enzyme signal. The stained bands then indictate the proteins to which the patient's serum contains antibody.