An Overview Of The Western Blot Immunoassay

Fri, 12/25/2009 - 10:00

Western blot is a widely used immunoassay technique, used to identify proteins. Many people enter Western blot research without having a clear idea of how the technique relates to antibody usage, so we at Novus Biologicals thought it would be interesting to give a general review of this subject.

Western blotting, also called protein immunoblotting, uses gel electrophoresis to separate proteins of tissue extracts and homogenates. Native and denatured proteins are identified by their isoelectric point, electric charge, molecular weight or molecular structure (often, a combination of these).

Tissue for Western Blot analysis is blended or homogenized to break down cells and release the proteins. Bacterial and viral proteins are also identified using Western blot, and samples are not restricted to cellular tissues. Various agents, such as buffers and inhibitors, are used to encourage cell lysis, solubilize proteins and inhibit their digestion by the sample’s own enzymes. Centrifugation and filtration is used to separate the cellular components.

Polypeptides are denatured with strong reducing agents to enable proteins to be separated by molecular weight. The most common type of gel electrophoresis used is SDS-PAGE. This uses Sodium Dodecyl Sulphate (SDS) to hold the proteins in their denatured state. The negatively charged SDS coats the proteins, which then move to a positively charged electrode through an acrylamide gel.

Once identified, individual proteins are transferred to a nitrocellulose or PVDF membrane, where they are detected using specific antibodies targeted to specific globulins. Antibody suppliers, such as Novus Biologicals, specialize in supplying monoclonal and polyclonal antibodies for a wide range of antigens, most of which are suitable for Western blot analysis.

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