Protein Pull-Down Techniques

Affinity GST Pull-Down Applications

Glutathione S-transferase (GST) is a 211 amino acid protein that is found in most organisms. GST is often integrated into expression vectors to produce the fusion tag within E. coli protein expression systems. The GST-tag is a large protein tag, approximately 26 kDa, and can be expressed in bacteria, yeast, mammalian, and insect cells. The GST-tag is advantageous when protection of the recombinant protein from intracellular protease cleavage is required, and when there is a need to enhance the solubility of the tagged protein. In addition, the GST protein provides a high-affinity tag for easy purification and for pull-down experiments utilizing inexpensive affinity resins that are performed in mild elution conditions.

Co-Immunoprecipitation

The pull-down assay is ideal for verification of co-immunoprecipitation results and an excellent method of identifying unknown protein-protein interactions along with the activation status of specific proteins. Unlike affinity pull-down methods, IP uses antibodies to bind to and isolate antigens from complex biological samples. 

Co-IP refers to the use of an antibody to bind an antigen within a multiprotein complex. The complex is then captured with the addition of protein A or protein G medium. The high affinity of protein A/G to the Fc region of polyclonal and monoclonal IgG-type antibodies makes them a critical component in the purification of the protein or protein complex of interest. The use of protein pull-down methods is an essential tool for investigating protein-protein interaction networks; however, the choice of the specific method will be determined by the researcher’s specific application need.