Protein Separation
Western blots are a biochemical technique that allow scientists to measure the presence and quantity of specific proteins. As MoelcularStation.com explains, the first part of a Western blot is separating out the proteins from the sample. These proteins can be obtained by breaking down (lysing) cells from a piece of tissue, or they can be obtained by taking them directly from the blood. Once the proteins have been obtained, the can be separated using a technique called electrophoresis, which separates proteins based on their size. The first part of electrophoresis is treating all of the proteins with a detergent, which helps straighten them out and also gives them an electrical charge. Next the proteins are injected into a gel which then has an electrical current passed through it lengthwise. The electrical current causes the proteins to move through the gel because they are electrically charged. Smaller proteins move faster through the gel, which allows the proteins to be dispersed throughout the gel based on size.
Membrane Transfer
Once the proteins have been spread out on the gel, they will form small horizontal bands that resemble rungs on a ladder. Thermo Fisher's Protein Methods Library explains that the next step is to get the bands of protein out of the gel and onto a membrane where they can be further analyzed. There are a number of ways of moving proteins from the gel onto the membrane, but the most commonly used one again involves an electrical current. In this case, the current is run at a 90 degree angle to the plane of the gel, which causes the proteins to move through the gel onto a membrane which is placed on top of the gel. This method is relatively quick and allows the proteins to be transferred while still keeping them separated based on their size.
Blotting
The "blot" portion of the Western blot utilizes antibodies. Antibodies are proteins that are produced by the immune system. Western blots use proteins that are specifically designed to only bind to the protein of interest (what the blot is attempting to detect). Usually, two different kinds of antibodies are used. The membrane is exposed to the first antibody (the primary antibody) which will then bind only to the protein of interest. The membrane is then washed off, leaving behind only the primary antibody that has bound to proteins on the membrane. Then the membrane is exposed to a second antibody (the secondary antibody) which binds to the primary antibody. This secondary antibody has a fluorescent molecule on one end which emits light. After any unbound secondary antibody is rinsed off, a piece of photographic film is put on the membrane. The fluorescent part of the secondary antibody will emit light, which will show up as a dark space on the film.
