![]() Approximately 500 ng of each of the purified proteins was separated by SDS-PAGE and stained with the Thermo Scientific Pierce Power Stainer (Cat. The additional bands (denoted with arrows) found with the purified proteins were previously identified by mass spectrometry as cellular proteins eEF1G and GSTM3, which are known to bind to the glutathione column and co-elute with GST tagged proteins. After binding to Thermo Scientific Pierce glutathione agarose, the GST-fused proteins were eluted with 50 mM glutathione. Genes cloned into pT7CFE1-NHis-GST-CHA were used to express GST-fusion proteins for 6 hours. Purification of N-terminal GST fusion proteins with immobilized glutathione. 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. 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. ![]() 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. The method of protein elution depends on the affinity ligand and ranges from using competitive analytes to low pH or reducing buffers.īesides investigating the interaction of two or more proteins, pull-down assays are a powerful tool to detect the activation status of specific proteins. 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 secondary affinity support of immobilized bait is then incubated with a protein source that contains putative "prey" proteins, such as a cell lysate. 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. 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. 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. 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. The pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins.
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