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