High-capacity, high-performance resins for GST-tagged fusion protein purification.
Thermo Scientific Pierce Glutathione Agarose is a high-capacity, high-performance resin for affinity purification of GST-tagged fusion proteins from cellular lysates.
The high-quality support consists of glutathione that has been immobilized by its central sulfhydryl group via a 12-atom spacer arm to crosslinked 6% beaded agarose. Purification of GST-fusion proteins using glutathione (GSH) agarose beads is well documented and adaptable to a variety of scales, column formats and specific applications. Whether the purpose is to purify large amounts of recombinant protein from over-expressing E. coli lysates or to investigate protein interactions involving GST-tagged bait proteins, Pierce Glutathione Agarose is suitable for the task.
To accommodate these many uses, Pierce Glutathione Agarose is offered in several package sizes and formats. These include three volumes of resin slurry, three sizes of centrifuge-ready columns, complete GST purification kits, two sizes of FPLC-ready chromatography cartridges, and 96-well filter plates for high through-put needs.
High capacity – binds at least 40mg of purified recombinant GST protein per milliliter of resin
High yield and purity – consistently purifies at least 10mg of GST-tagged protein per milliliter of resin with greater than 90% purity
Cost-effective – resin is economically priced and can be reused at least five times without reduction in binding capacity and purification performance
Versatile – works well to purify GST-fusion proteins from bacterial lysates or use with pre-purified GST-tagged proteins to pull down protein interactions
Compatible – validated and effective for use with Thermo Scientific Cell Lysis Reagents to extract and purify from bacterial or mammalian cell cultures
Flexible – available in multiple formats including bulk resin, spin columns, chromatography cartridges, 96-well filter plates and convenient GST purification kits
Chemical structure of immobilized GSH (Thermo Scientific Pierce Glutathione Agarose).
Pierce Glutathione Agarose effectively purifies high levels of overexpressed GST-tagged fusion proteins from bacterial lysates, such as those that are obtained with Thermo Scientific B-PER Bacterial Protein Extraction Reagents.
High quality purification of different GST fusion proteins using Thermo Scientific Pierce Glutathione Agarose. Three GST-fusion proteins were expressed in E. coli, extracted in Thermo Scientific B-PER Reagent with Enzymes (Part No. 90078), and then purified using a Pierce Glutathione Agarose product. Elution fractions were separated on 4-20% gradient Tris-glycine gels and stained with Thermo Scientific GelCode Blue Stain Reagent (Part No. 24590).
Pak1-GST: Lysate (2.4mg total protein) was purified using the Pierce GST Spin Purification Kit (Part No.16106). Crude lysate was applied in Equilibration-Wash Buffer to a 0.2mL Pierce Glutathione Spin Column and then eluted with 10mM Glutathione Elution Buffer, pH 8.0. Yield of target Pak1-GST was 475µg.
Bid-GST: Expressing cells (225mg pellet) were lysed and the extract (11mg total protein) was applied to a 0.2mL Pierce Glutathione Spin Column (Part No. 16103) and eluted with 125mM Tris, 150mM NaCl, 10mM Glutathione, pH 8.0. Yield of target Bid-GST was 3.63mg.
ERK-GST: Expressing cells (480mg pellet) were lysed and 3mL cell extract (11.2 mg total protein) was applied in 50mM Tris, 150mM NaCl, pH 8.0 buffer to a column containing 0.5mL of Pierce Glutathione Agarose (Part No. 16100) and eluted with 125mM Tris, 150mM NaCl, 10mM Glutathione, pH 8.0. Yield of target Erk-GST was 1.132mg.
Pierce Glutathione Agarose performs well in batch-binding and spin-column procedures at a variety of scales. Performance equals or exceeds popular GSH resins from other suppliers.
Thermo Scientific Pierce Glutathione Agarose delivers high yield and high purity GST-fusion proteins.E. coli lysate (14.4mg total protein) containing overexpressed GST was incubated with 50µL GSH resin from various suppliers and purified per manufacturers' instructions. The amount of GST eluted from the resin (yield) was quantified by Thermo Scientific Coomassie Plus Protein Assay. Purity was assessed by densitometry of the stained gel lanes. M= MW marker; L=Lysate load; FT=Flow-through; E=Elution.
Pierce Glutathione Agarose is a high-quality, stable and resilient affinity support. Tests confirm that no decrease in performance occurs after at least five repeated uses. These data indicated that the resin is highly resistant to structural degradation or ligand leaching during normal use.
Reuse Thermo Scientific Pierce Glutathione Agarose without loss in protein purification efficiency. Stained polyacrylamide gel (SDS-PAGE) of fractions from five successive rounds of purification with one aliquot of resin. Each round consisted of incubation of E. coli lysate (1.5mg total protein) over-expressing GST with 50µL Pierce Glutathione Agarose in a spin column. The flow-through (left lane in each group) and subsequent wash (middle lane) fractions were recovered, then the GST protein was eluted (right lane) with 125mM Tris, 150mM sodium chloride and 10mM glutathione. Finally, the resin was treated between each use with regeneration buffers.
The expression and purification of recombinant proteins is central to protein regulation, structure and function studies. The majority of recombinant proteins are expressed as fusions with short affinity tags or small proteins, such as glutathione S-transferase (GST). This protein binds specifically to reduced glutathione (GSH) in near-neutral, nondenaturing conditions (e.g., Tris buffer). Bound protein is easily dissociated (eluted) by competitive displacement with buffer containing free, reduced GSH (oxidized glutathione, GSSH is not effective for this purpose). When proteins of interest are expressed as fusions with GST and glutathione is immobilized to an solid support, this protein-substrate system enables affinity purification of recombinant proteins, as well as various other experiments with those proteins.