GST Pull Down Protein Interaction Complex Protein Expression And Purification

Introduction to pull-down assays

The pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. Pull-down assays are useful for both confirming the existence of a protein–protein interaction predicted by other research techniques (e.g., co-immunoprecipitation) and as an initial screening assay for identifying previously unknown protein–protein interactions.

Pull-down assays are a form of affinity purification and are similar to immunoprecipitation, except that a "bait" protein is used instead of an antibody. Affinity chromatography (i.e., affinity purification) methodologies greatly enhance the speed and efficiency of protein purification and simultaneously provide the technology platform to perform a pull-down, or co-purification, of potential binding partners. In a pull-down assay, a bait protein is tagged and captured on an immobilized affinity ligand specific for the tag, thereby generating a "secondary affinity support"’ for purifying other proteins that interact with the bait protein. The secondary affinity support of immobilized bait is then incubated with a protein source that contains putative "prey" proteins, such as a cell lysate. The source of prey protein at this step depends on whether the researcher is confirming a previously suspected protein–protein interaction or identifying an unknown interaction. The method of protein elution depends on the affinity ligand and ranges from using competitive analytes to low pH or reducing buffers.

Besides investigating the interaction of two or more proteins, pull-down assays are a powerful tool to detect the activation status of specific proteins. For example, proteins that are activated in response to tyrosine phosphorylation can be pulled down using an immobilized SH2 domain that targets the phosphorylated tyrosine on a given protein. Additionally, GTPases, which act as molecular switches that regulate cell signaling by cycling between a GTP-bound (active) and GDP-bound (inactive) state, can be pulled down using an immobilized GTPase-binding domain of downstream proteins that are recruited to GTP-bound, activated GTPases. In both types of pull-down assays, because the specificity of the interaction is dependent on the sequence of the binding domain, these approaches are highly specific in detecting the activation of distinct proteins.

Advantages and disadvantages with different pull-down assays for the retrieval of protein complexes