Western blot

Western blot (also called immunoblot) is a technique to detect specifically one protein in a mixture of large number of proteins and to obtain information about the size and relative amounts of the protein present in different samples.

In this method first proteins are separated using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE).

Then they are moved onto a nitrocellulose membrane. The proteins retain the same pattern of separation they had on the gel.

The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. This step is required as both antibodies and the target are proteins and proteins like to bind to nitrocellulose. By placing the nitrocellulose membrane in a dilute solution of protein, we can prevent the unwanted interactions between the membrane and the antibody used for detection of the target protein as the protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached.

An antibody is then added to the solution which is able to bind to its specific protein and forms an antibody-protein complex with the protein of interest. (In fact there is no room on the membrane for the antibody to attach other than on the binding sites of the specific target protein).

Finally the nitrocellulose membrane is incubated with a secondary antibody, which is an antibody-enzyme conjugate that is directed against the primary antibody.

The location of the antibody is revealed by incubating it with a substrate that the attached enzyme converts to a product that can be seen and followed and then photographed.




Western blot is a confirmatory test for a positive HIV ELISA as it allows one to visualize antibodies directed against different viral protein in a human serum sample.