FLAG-Fusion Protein Purification

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).