GE Healthcare’s Glutathione Sepharose 4B is an affinity chromatography medium which has affinity for GST and other glutathione binding proteins. It can be used to purify GST-tagged recombinant proteins expressed from bacterial cell lysates which contain protein expressed the pGEX series of expression vectors. Its total binding capacity is 10 mg recombinant GST/ml medium.
Glutathione Sepharose 4B comes as a 60% slurry in 20% ethanol; a 50% slurry should be prepared before use. It is to be stored at 4-8ºC. The other reagents required for purification are routinely-used chemicals. To prepare the 50% slurry, 1.3 ml of the 60% slurry is placed in a 15 ml tube and 10 ml of chilled PBS is added into it, mixed gently and kept upright at 4ºC for overnight. The following morning, the upper solution is decanted and 10 ml of chilled PBS is added again, mixed gently and centrifuged at 1000 rpm for 5 min at 4ºC. The upper solution is again removed and 1 ml of PBS is added, you then have a 50% slurry. Add 150 ul of this slurry to 275 ug of GST in PBS with 1% Triton X-100 in a 15 ml tube; the amount of GST is estimated given that the typical yield from a pGEX vector is 2.5 ug GST fusion protein/ml culture. We use this ratio of slurry to GST knowing that 2 ml of a 50% slurry can bind 5 mg of GST protein. After the slurry has been added to the lysate, it is kept on rocker at 4ºC for 1h and then centrifuged at 1000 rpm at 4ºC for 5 min. The supernatant is then discarded, but approximately 500 ul should be left on top of the beads. The beads and the residual supernatant are transferred to a microcentrifuge tube. The beads are then washed 3 times with chilled PBS. After removing PBS, 500 ul of elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0) is added and kept on rocker at 4ºC for 10 min. It is then centrifuged at 1000 rpm at 4ºC for 5 min, the supernatant is stored and the elution is repeated for 4-5 times. These eluates can be pooled together and analyzed by SDS-PAGE.
My research aims to identify proteins/tumor antigens which elicit an antibody response in cancer. We have identified an autoantibody response against a few tumor antigens in the sera of cancer patients. To confirm the autoantibody response, recombinant proteins of these tumor antigens are prepared and analyzed. These recombinant proteins are expressed using the pGEX-4T1 vector in BL21 cells. In order to purify GST fusion proteins from the bacterial cell lysate, Glutathione Sepharose 4B is used and a significant amount of pure protein is recovered. The purified GST-fusion proteins yielded good results in screening for antibodies in the sera of the patients.
I, therefore, recommend Glutathione Sepharose 4B for quick and easy purification of recombinant GST fusion proteins.