Isolation and analysis of GST-tagged proteins
Background The glutathione S-transferase (GST) enzyme family comprises a long list of cytosolic, mitochondrial, and microsomal proteins which are capable of multiple reactions with a multitude of substrates, e.g. glutathione. Genetic engineers have used glutathione S-transferase to create the so-called GST gene fusion system. Here, the GST tag is used to purify and detect glutathione-binding proteins of interest. A high-quality monoclonal Anti-GST antibody coupled to µMACS™ MicroBeads is offered by Miltenyi Biotec, allowing specific isolation of recombinant GST fusion proteins on an analytical scale. For medium- to large-scale purification of GST-tagged proteins, the ProCatch Glutathione Resin is recommended (fig. 1). Isolation and purification of GST-tagged proteins The µMACS™ and MultiMACS™ GST Isolation Kit take advantage of the well-established MACS® Technology; thus, allows fast and specific isolation of pure GST-tagged proteins. The advantages of MACS Technology for protein isolation are: Specificity – high-quality monoclonal antibodies coupled to MACS MicroBeads assure specific isolation of GST-tagged proteins with low background. Sensitivity – fast reaction kinetics of tiny (50 nm), non-sedimenting MACS MicroBeads allow sensitive isolation even when working with rare proteins. Speed – less than 2 hours to high-purity GST-tagged proteins! The µMACS GST Isolation Kit was developed for manual, low-throughput applications with the µMACS Separator. The procedure can easily be upscaled with the MultiMACS GST Isolation Kits to a semi- or fully-automated, high-throughput processing of up to 96 samples in parallel by utilizing the MultiMACS 96 Separator. For medium- to large-scale purification of GST-tagged proteins the ProCatch Glutathione Resin is recommended. Antibody specifications Target sequence: whole glutathione S-transferase (220-residue polypeptide) Tag origin: Schistosoma japonicum Isotype: mouse IgG1 Columns For µMACS™ Isolation Kit: µ Column, upscale with M Column For MultiMACS™ Isolation Kits: Multi-8 or Multi-96 Columns Further information Protein isolation flyer.pdf [PDF; 814,4 KB]		Figure 1 Purification of a 30 kDa glutathione-binding protein with ProCatch Glutathione Resin. A Marker (lane 1), lysate from cell extract (2), flow-through (3), first eluate (4), and second eluate (5) were separated by SDS-PAGE and Coomassie stained. B Low-pressure column purification protocol. Peak 1 defines non-bound protein fraction (flow-through). Peak 2 corresponds to the eluted glutathione-binding protein. Absorbance was measured at 280 nm. A B

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