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Glutathione-S-transferase (GST) fusion proteins have had a range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria1. Typically, GST pull-down experiments are used to identify interactions between a probe protein and unknown targets and to confirm suspected interactions between a probe protein and a known protein2,3. The probe protein is a GST fusion, whose coding sequence is cloned into an isopropyl-β-D-thiogalactoside (IPTG)-inducible expression vector. This fusion protein is expressed in bacteria and purified by affinity chromatography on glutathione-agarose beads. Target proteins are usually lysates of cells, either labeled with [35S]methionine or unlabeled, depending on the method used to assay the interaction between the target and the probe. The cell lysate and the GST fusion protein are incubated together with glutathione-agarose beads. Complexes recovered from the beads are resolved by SDS-PAGE and analyzed by western blotting, autoradiography or staining.
Beyond protein destruction: a new real-time assay to measure dynamic changes in ubiquitination promises to highlight the many other roles of this critical protein modification.
The DNA marketplace is changing: the technology is maturing, whole human genome arrays, as well as those of model organisms, are beginning to make it onto the market, and researchers have more choices of commercial platform than ever before. Diane Gershon takes a closer look at what's on offer.
