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Protein Expression & Purification


Products >  Protein_Expression_and_Purification >  Protein_Learning_Center >  Tagged_Protein_Purification >  GST-Tagged_Protein_Purification_Overview

GST-Tagged Protein Purification Overview

Glutathione-Superflow Resin allows rapid affinity purification of GST-tagged proteins for batch, gravity flow, and FPLC applications. The resin is based on 6% cross-linked agarose, with glutathione covalently bound to the resin. It can withstand flow rates as high as 15 ml/cm2/min and medium pressure (up to 150 psi).

Applications

Epitope tags such as glutathione-S-transferase (GST) are often used to label proteins for expression and purification applications. Glutathione transferases are abundant enzymes involved in cellular defense against electrophilic chemical compounds, which bind glutathione with high affinity and specificity. The strength and selectivity of this interaction can be exploited to effectively purify GST-tagged proteins. The Glutathione-Superflow Resin selectively binds the GST-tagged protein under normal conditions, allowing the protein of interest to be separated from whole cell extracts rapidly and efficiently. A high degree of purification can be achieved in just one chromatographic step.

GST-tagged protein purification from whole cell extracts.

GST-tagged protein purification from whole cell extracts using Glutathione-Superflow Resin. Whole cell extracts containing GST-DHFR (Panel A) and GST alone (Panel B) were loaded, washed and eluted from Glutathione-Superflow Resin columns. Then, the resulting purification fractions were analyzed by SDS-PAGE (upper panels) and Western blot (lower panels) with an anti-GST IgG.

GST is a 35 kDa protein that can be assayed biochemically as well as immunologically. This characteristic sets it apart from many epitope tags that are simply short peptides. However, since GST is a large tag, the potential for degradation by proteases is higher than for smaller tags. Therefore, performing GST-based protein purification as quickly as possible under non-degrading conditions is necessary to minimize sample loss. GST loses its ability to bind Glutathione-Superflow Resin when denatured, and consequently strong denaturants such as guanidine-HCl or urea must not be used in the purification buffers. Check reagent compatibilities when designing your purification scheme.

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