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.
