It binds to the first antibody, and is usually produced by a different animal. Some high affinity monoclonal antibodies can also be used for Western blots.Īfter rinsing the membrane to remove unbound primary antibody a secondary antibody is incubated with 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 body) and collecting the antibodies the animal produces against that protein. The primary antibody recognizes only the protein of interest, and will not bind any of the other proteins on the membrane. The diluted antibody solution and the membrane can be sealed in a plastic bag and gently agitated for an "incubation" of about half an hour. The antibody is diluted in a solution containing a modest amount of a salt such as sodium chloride, some protein (such as BSA) to prevent non-specific binding of the antibody to surfaces and a small amount of a buffer to keep the solution near neutral pH. The first antibody (often called the primary antibody) is incubated with the membrane. Without the blocking, the antibody to be applied in the next step would bind to the nitrocellulose. This is done by placing the membrane in a solution of Bovine serum albumin (BSA) or non-fat dry milk. The membrane is then blocked, in order to prevent non-specific protein interactions between the membrane and the antibody protein (next step, below). Unlike nitrocellulose, PVDF must be soaked in 100% methanol before using. PVDF is often used because it is sturdier and can be "stripped" of antibodies and reused. Protein binding is based upon hydrophobic interactions as well as charged interactions between the membrane and protein. The membrane is "sticky" and binds proteins non-specifically (i.e. This is the actual blotting process and is necessary in order to expose the proteins to antibody (see below). The proteins in the gel are then transferred onto a membrane made of nitrocellulose or PVDF, by pressure or by applying a current. However, it is also possible to use a 2-D gel which spreads the proteins from a single sample out in two dimensions. Usually the gel has several lanes so that several samples can be tested simultaneously. The proteins of the sample are separated according to size on a gel, usually using SDS-PAGE. Detection of RNA is termed Northern blotting. Its name is a pun off the name Southern blot, a technique for DNA detection developed earlier by Edward Southern. It also gives information about the size of that protein. Picture of a Western blot with 5 vertical lanesĪ Western blot is a method in molecular biology to detect a certain protein in a sample by using antibody specific to that protein.
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