As previously described (31 (link)), cells were lysed with 1 × RIPA lysis buffer (P0013B, Beyotime) for 30 minutes at 4°C. Glutathione S-transferase (GST) fusion proteins were immobilized on BeyoMag Anti-GST Magnetic Beads (P2138, Beyotime). After washing with 1 × RIPA lysis buffer, the beads were incubated with cell lysates for 4 hours. The beads were then washed four times with 1 × RIPA lysis buffer and resuspended in loading buffer. The bound proteins were subjected to SDS/PAGE and Western blotting.
Escherichia coli BL21 was used to express GST-recombinant proteins and His-recombinant proteins after induction with IPTG (Beyotime). Then Escherichia coli BL21 was lysed with muramidase and sonication. Bacterial debris was removed by centrifugation for 10 minutes. Glutathione-Sepharose beads (GE Healthcare Life Sciences) were added to the liquid supernatant to purify GST-fusion proteins at 4°C overnight. The beads were then collected and washed for six times with binding buffer to remove the contaminating proteins. SDS-PAGE and high-sensitivity colloidal Coomassie blue staining were performed to examine the purification efficiency. For purification of His-tagged proteins, HisSep Ni-NTA MagBeads (Yeasen, 20561ES03) were used to isolate and purify His-tagged proteins. The remaining steps were similar to those for Glutathione-Sepharose beads.
Escherichia coli BL21 was used to express GST-recombinant proteins and His-recombinant proteins after induction with IPTG (Beyotime). Then Escherichia coli BL21 was lysed with muramidase and sonication. Bacterial debris was removed by centrifugation for 10 minutes. Glutathione-Sepharose beads (GE Healthcare Life Sciences) were added to the liquid supernatant to purify GST-fusion proteins at 4°C overnight. The beads were then collected and washed for six times with binding buffer to remove the contaminating proteins. SDS-PAGE and high-sensitivity colloidal Coomassie blue staining were performed to examine the purification efficiency. For purification of His-tagged proteins, HisSep Ni-NTA MagBeads (Yeasen, 20561ES03) were used to isolate and purify His-tagged proteins. The remaining steps were similar to those for Glutathione-Sepharose beads.
