Western blotting is a common tool used for detecting proteins in crude protein mixtures such as serum and cell lysate. The proteins in the mixture are separated on the basis of size and charge by gel electrophoresis and then transferred onto nitrocellulose. This provides a solid matrix with immobilized protein. The separated proteins are then “probed” with specific antibodies. Bound antibodies are detected using a secondary antibody conjugated to an enzyme, similar to an ELISA assay. Chromogenic substrates include those where the products precipitate on the nitrocellulose or chemiluminesce that can be detected using autoradiography.
- Antigen detection and quantitation in complex samples
- Cross-reaction with homologous antigens
- Binding comparison of different antibodies to single antigens
- Antibody binding to antigen fragments and chemically modified antigens
Some important points about western blots:
- Not all monoclonal antibodies can bind proteins following reduction of disulfide bonds and denaturation with sodium dodecylsulfate.
- Antibodies recognizing linear, or non-conformation dependent epitopes are more likely useful for immunohistochemistry or other assays where antigens are subjected to denaturing conditions.
- Cellular components can be identified from cell lysates or membrane preparations
- Proteins can be enriched by immunoprecipitation prior to gel electrophoresis.
- Antibodies that bind well in western blots do not always bind strongly in ELISA when the antigen is immobilized on plastic.
- In order to obtain monoclonal antibodies that bind proteins in western blot assays, positive antibodies from ELISA should be screened directly in western blots early in the hybridoma selection process.