Pull-Down Assay

Pull-Down assay is an in vitro method used to determine a physical interaction between proteins. It can be used to confirm a protein-protein interaction discovered through other techniques, and to explore previously unknown protein-protein interactions of specific proteins.

Pull-Down assay is quite similar to immunoprecipitation(IP), except that a bait protein replaces an antibody as affinity reagent. Pull-Down assay can greatly enhance the speed and efficiency of protein purification and exploration of potential binding partners, especially the ones stably bound to bait protein . In a Pull-Down assay, the bait proteins are usually tagged with GST, polyHis, or biotin, and captured on an immobilized affinity ligand against the tags, such as glutathione nickel column or streptavidin, and thereby generating a "secondary affinity support"' for isolating proteins bound to bait proteins. The secondary affinity support of immobilized bait is then incubated within a biological sample, to capture putative prey proteins. And then protein complex would be washed to eliminate unspecific binding. Identification of bait-prey interactions requires that the complex is removed from the affinity support and analyzed by detection methods, such as SDS-PAGE. But SDS-PAGE loading buffer is harsh which will denature all proteins in the sample. And competitive analyte elution is much more specific for the bait-prey interaction.

Besides confirmation and discovery of interactions between proteins, Pull-Down assay is also a powerful tool to detect the activation status of specific proteins. For example, proteins activated in response to tyrosine phosphorylation, can be pulled down with an immobilized SH2 domain which targets the phosphorylated tyrosine on a given protein. And this method is highly specific to detect whether distinct proteins are activated. The tech team of Creative Proteomics can have provide professional analytical support to the customers after having a detailed conversation with you.

http://www.creative-proteomics.com/services/pull-down-assay.htm