Protein Pull-Down
The pull-down assay is a technique designed to investigate the interaction between two or more proteins. In affinity pull-down assays, a 'bait' protein is tagged and captured on an immobilized ligand, such as support beads, either through covalent attachment or via an affinity tag, like immobilized metal affinity chromatography (IMAC). Common bait protein fusion tags include Glutathione S-transferase (GST) and histidine-tagged proteins. The bait protein forms a complex, which is then incubated with a protein source, such as a cell or tissue lysate. After incubation, the proteins of interest are eluted from the support beads through a series of washes and collected by centrifugation. The affinity pull-down method of purification and detection differs from immunoprecipitation (IP) and Co-IP assays in that it does not rely on antibody-antigen interactions. For further downstream analysis, the purified sample is often analyzed using techniques such as SDS-PAGE, mass spectrometry, and western blot detection,
Overview
Affinity GST Pull-Down Applications
Glutathione S-transferase (GST) is a 211 amino acid protein commonly used as a fusion tag in protein expression systems. The GST-tag, approximately 26 kDa in size, is beneficial for protecting recombinant proteins from protease cleavage and enhancing protein solubility. GST also provides a high-affinity tag for easy purification and pull-down experiments using inexpensive affinity resins. This method is particularly advantageous when working with mild elution conditions, making it a versatile tool for protein purification and interaction studies in E. coli, yeast, mammalian, and insect cells.
Co-Immunoprecipitation
Co-immunoprecipitation (Co-IP) is a powerful method for verifying protein-protein interactions, often used to identify unknown interactions or the activation status of specific proteins. In Co-IP, antibodies bind to an antigen in a multiprotein complex, which is then captured using protein A or protein G medium. The high affinity of protein A/G to the Fc region of IgG antibodies enables effective isolation of the protein or complex of interest. This method is essential for investigating protein interaction networks and is commonly used in conjunction with other pull-down techniques depending on the research needs.