| Definition |
Proteins are fractionated by PAGE (SDS-polyacrylamide gel electrophoresis), transferred to a nitrocellulose membrane and tested for the ability to bind to a protein, a peptide, or any other ligand. Cell lysates can also be fractionated before gel electrophoresis to increase the sensitivity of the method for detecting interactions with rare proteins. Denaturants are removed during the blotting procedure, which allows many proteins to recover (or partially recover) activity. However, if biological activity is not recoverable, the proteins can be fractionated by a non denaturing gel system. This variation of the method eliminates the problem of activity regeneration and allows the detection of binding when the presence of a protein complex is required for binding. The protein probe can be prepared by any one of several procedures, while fusion affinity tags greatly facilitate purification. Synthesis in E. coli with a GST fusion, epitope tag, or other affinity tag is most commonly used. The protein of interest can then be radioactively labelled, biotinylated, or used in the blotting procedure as an unlabeled probe that is detected by a specific antibody.[ PubMed:7708014 ] |
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