| Popis: |
Publisher Summary This chapter presents the detailed methods for Western blotting, with pertinent examples relevant to oncogene research. Western blotting is a versatile and widely used technique in oncogene research. This method relies on transfer of proteins out of a polyacrylamide gel matrix and onto the surface of a filter. L2 Several filter matrices are available which adsorb proteins on their surface, most likely through hydrophobic interaction, without the need for covalent cross-linking. Once immobilized on a solid support, the adsorbed proteins are readily accessible to added ligands and can be used to detect antigen-antibody, protein-protein, or DNA/RNA-protein interactions. The first step in a Western blot procedure is typically denaturing SDS-PAGE. Samples for transfer are usually prepared in SDS-PAGE sample buffer. For 2Delectrophoresis, samples are prepared in sample buffer for isoelectric focusing; the second dimension SDS-PAGE gel is then blotted. Transfer of proteins electrophoresed through native gels has also been reported. |
