Regeneration and Storage of Glutathione Resin
Glutathione Resin can be reused to purify the same protein three times without regeneration. If the target GST-fusion protein is different, however, the Glutathione Resin must be regenerated using the following protocol:
1. Wash the column with 2×bed volumes of 0.1 M Tris HCl + 0.5 M NaCl, pH 8.5.
2. Wash the column with 2×bed volumes of 0.1 M sodium acetate + 0.5 M NaCl, pH 4.5.
3. Re-equilibrate the column with 3-5×bed volumes of 1×PBS.
4. For long-term storage, the resin should be stored in 1×PBS containing 20% ethanol at 2 - 8°C.

Preparation of Cell Extract
1.Harvest cells by centrifugation at 3,000 g at 4°C for 10 min, remove and discard the supernatant.
2. Resuspend the cell pellet in 3 ml ice-cold 1×PBS buffer per 50 ml culture and centrifuge at 3,000 g at 4°C for 10 min. Remove and discard the supernatant.
3. Freeze the cell pellet at -80°C for 1 hour (This is also a convenient point to stop and one can continue the procedure later).
4. Thaw cell pellet on ice and resuspend cells in 3 ml of ice-cold 1×PBS buffer per 50 ml culture. If desired, add appropriate additives, such as non-ionic detergents (NP-40) or protease inhibitors (PMSF).
5. Disrupt cells by brief pulses of sonication on ice until the sample is no longer viscous.
6. Centrifuge at 12,000 g at 4°C for 10 min and carefully transfer the supernatant (soluble fraction) to a clean and pre-chilled tube and resuspend pellet (insoluble fraction) with 3 ml of ice-cold 1×PBS buffer per 50 ml culture.
7. Aliquot 10 μl samples from both soluble and insoluble fractions for SDS-PAGE analysis [ by adding equal volume of 2X SDS Sample Buffer (125 mM Tris-HCl, pH 6.8, 4% w/v SDS, 20% glycerol, 100 mM DTT,0.02% w/v bromophenol blue), boiling for 5 min and running SDS-PAGE to determine the amount and solubility of the GST-fusion protein].

Note:
1. The binding of GST or GST-fusion protein to Glutathione Resin is not affected by 1% Triton X-100, 1% Tween-20, 1% CTAB, 10 mM DTT, 0.03% SDS, or 0.1% NP-40. These chemicals may be used to reduce non-specific binding.
2. If the target GST-fusion protein forms inclusion body (insoluble protein), the inclusion body has to be properly solubilized and refolded prior to purification.

Purification of Recombinant GST-Fusion Protein
1. Completely resuspend the Glutathione Resin by gently shaking the vial.
2. Transfer an appropriate amount of slurry to a disposable column (included in Kit L00207 and L00208). Usually 1 ml settled resin (2 ml 50% slurry) can bind more than 6 mg horse liver GST protein.
3. Wash the Glutathione Resin with 10×bed volumes of cold (4°C) 1×PBS.

1. Apply clear solution (sonicate, etc) containing GST-fusion protein in cold 1×PBS to the equilibrated column with the flow rate at 10-15 cm/h.
   5. Add 1×PBS to wash the column just after all the protein solution get into the column, use 20×bed volumes of PBS for wash. Protease inhibitors such as PMSF are better added to wash solution to inhibit protease activity.
   6. Elute the fusion protein with 10-15×bed volumes of freshly made 10 mM glutathione elution buffer (0.154 g of reduced glutathione dissolved in 50 ml of 50 mM Tris-HCl, pH 8.0.).
   7. Monitor elution of the fusion protein using absorbance readings at 280 nm.
   8. Aliquot 10-20 μl supernatant containing GST-fusion protein, flow-through, wash and the eluted protein, respectively, and analyze all the samples by running SDS-PAGE to confirm the presence of the target protein.
   9. Pool eluted fractions containing target protein. Remove free glutathione by dialysis at 4°C against a buffer of choice or by using a G15 Sephadex desalt column.