Analysis of Protein Interactions in Vitro

Protein-protein interactions can be detected many ways in vitro. Some of these include coimmunoprecipitation, protein pulldown assay, chemical crosslinking, fluorescence resonance energy transfer (FRET), label transfer, and tandem affinity purification (TAP). Of these, TAP is the only high-throughput method. 

The coimmunoprecipitation of proteins by an antibody is the standard procedure to demonstrate the presence of interacting proteins. In this method, when a protein is precipitated by a specific antibody, the interacting protein(s) are also precipitated along with the protein precipitated by the antigen-antibody interaction the components of interacting protein are then visualized by Western analysis within the same band on the gel after electrophoresis. 

Protein pulldown works like coimmunoprecipitation except for the use of a ligand instead of an antibody to detect the interacting proteins on gel electrophoresis. The combination of the interacting proteins is observed to move much slower on the gel than the individual proteins in the interacting group. A label transfer is used to detect the proteins with a weak or transient 

Mutation that destroys DNA binding domain (BD) of Gal4p 

GAL4 transcription activation ( Translation doman mutant ----- 

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