Large scale FPLC purification of GST produces >95% purity of target protein

Biomass (170g) containing overexpressed GST was lysed with 1.7L of lysis buffer and then 0.75L of lysate was loaded onto equilibrated 200mL columns (50mm x 100mm) of Thermo Scientific Pierce Glutathione Superflow Agarose (left) or Glutathione Sepharose™ 4 Fast Flow (GE Healthcare, right) at a linear flow rate of 30cm/hour. Columns were washed with binding buffer until the UV280 reached baseline; then bound protein was eluted with elution buffer, and fractions containing purified GST were pooled. Load, flow-through, wash, and eluate fractions were separated by SDS-PAGE, stained with Imperial Protein Stain (Part No. 24615) and evaluated using myImageAnalysis Software (Part No. 62237) to determine purity. Total yield, recovery, and purity were nearly identical for both resins.

Biomass (170g) containing overexpressed GST was lysed with 1.7L of lysis buffer and then 0.75L of lysate was loaded onto equilibrated 200mL columns (50mm x 100mm) of Thermo Scientific Pierce Glutathione Superflow Agarose (left) or Glutathione Sepharose™ 4 Fast Flow (GE Healthcare, right) at a linear flow rate of 30cm/hour. Columns were washed with binding buffer until the UV280 reached baseline; then bound protein was eluted with elution buffer, and fractions containing purified GST were pooled. Load, flow-through, wash, and eluate fractions were separated by SDS-PAGE, stained with Imperial Protein Stain (Part No. 24615) and evaluated using myImageAnalysis Software (Part No. 62237) to determine purity. Total yield, recovery, and purity were nearly identical for both resins.

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