Novus Biologicals | Antibody News

Western blotting combines gel electrophoresis with use of a membrane to separate and identify target proteins using antibodies. Proteins are separated into bands using electrophoresis, and are then transferred to a membrane using filter-paper capillary action or an electroblotting technique. The effectiveness of the transfer can be checked by means of a stain – typically Ponceau S.

The membrane is then blocked from further protein interaction with diluted bovine serum albumin or non-fat milk solution. This ensures the antibodies bind only to the proteins, and not to the membrane substrate. False positives and fuzzy results are thus minimized, giving coherent and repeatable results.

The next stage is to incubate the membrane with a diluted primary antibody solution – typically 0.5 to 5.0 mcg/mL. The membrane is rinsed to remove any unbound protein, and exposed to a secondary antibody. Primary immunoglobulins are species derived, and secondary antibodies target to a species-specific portion of the primary agent. An anti-murine (mouse) secondary, for example, will bind to all mouse-sourced primaries.

This enables labs to share primary proteins in mass quantities, producing consistency between results and reducing costs.

Secondary antibodies are linked to a biotin or reporter enzyme, such as horseradish peroxidase. When the enzyme interacts with the relevant substrate, a color-reaction agent is activated which visualizes the protein on the membrane. Other detection methods include chemoluminescence, where an agent luminesces in proportion to the amount of protein present and fluorescent dyes activated by near infra-red bandwidths of light. The latter is considered the most precise method.

We at Novus Biologicals have a huge antibody database, including the latest fluorescent products.

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.