Electrophoresis
The first step of analyzing blood via a western blot is to separate out the proteins in the blood. Western blots work by detecting the presence of proteins. The blood is filled with proteins, but they differ in size. As a result they can be separated via a procedure called gel electrophoresis. As WesternBlotting.org explains, gel electrophoresis involves injecting a small amount of the blood into a gel. The proteins are then exposed to a detergent called SDS, which helps unfold the proteins and makes them mildly electrically charged. When an electric current is applied along the length of the gel, the proteins will begin to move through the gel. Smaller proteins will move through the gel faster, which allows the proteins to then be separated based on their size. The proteins will then separate out into small horizontal bands based on their relative size.
Transfer
According to Lab Tests Online, once the proteins have been adequately separated, the next step is to transfer the proteins onto a membrane. This thin membrane will serve as the base for the "blotting" portion of the test. Transferring the separated proteins from the gel to the membrane again involves the administration of an electric current, but this time the current is applied at a ninety degree angle to the gel so that the proteins move from the gel onto the membrane. The proteins bind to the membrane and are held in place.
Blotting
The final part of a western blot involves the actual blotting procedure, which uses antibodies. Antibodies are proteins made by the immune system. They are manufactured to bind to the protein that is being tested for. When applied to the membrane at the right concentration, they will bind only to the protein of interest. Most western blots actually use two different antibodies: one that binds to the protein of interest (the primary antibody) and another which binds to the primary antibody (the secondary antibody). The secondary antibody has a molecule that can emit fluorescent light. The membrane is washed (so that any unbound antibodies are washed away) and then developed by placing a piece of photographic film against the membrane. The fluorescent secondary antibody will emit light that will be detected as a dark band on the photographic film, which then demonstrates the presence of the protein of interest in the blood.
