Glutathione resin

Protein purification followed the procedures described by Fu et al. (2017) (link). Plasmids expressing FLAG-fusion proteins were transfected into the HEK293T cell line. Transfected cells were lysed using Pierce IP lysis buffer (ThermoFisher Scientific; TFS) with protease inhibitor cocktail (Roche). After cell debris was removed by centrifugation, anti-FLAG M2 affinity gel beads were added to cell extracts and incubated for 16 hr at 4°C. Beads-FLAG-protein complex was eluted by incubation with 3X FLAG peptide for one hr. The FLAG-fusion proteins eluate was restored and used for the following experiment. Plasmids pGEX-GST and pGEX-GST-Nogo66 were used to express recombinant proteins using 0.1 mM Isopropyl β-D-1-thiogalactopyranoside induction for 1 hr at 37°C in an Escherichia coli BL21 (ATCC BAA-1025TM) expression system and purified by Glutathione resin (Clontech).

GST-tagged Annexin A6 in pGEX-4T-1 vector was used for bacterial expression in BL21 E. coli strain. GST fusion protein was isolated using glutathione resin (Clontech, USA) [17 (link)] and stored as 50% glycerol slurry. pCDNA(−)-HA-EGFR transfected HEK293T cells were washed with PBS and incubated on ice for 15 min with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, and 10% glycerol). Lysate was clarified by centrifugation and incubated with glutathione resin loaded with GST-Annexin A6 (2 h, 4 °C). The resin was then collected by centrifugation and washed three times with lysis buffer, and the amount of HA-EGFR bound to Annexin A6 beads was detected by western blot analysis.