Purification of TDP-43 Recombinant Proteins

Recombinant TDP-43 proteins were purified by following previously described procedures^{43 (link)}. For purification of full-length TDP-43–MBP–6×His, TDP-43-5FL–MBP–His, TDP-43-5FL–eGFP–MBP–His and TDP-43-ΔLCD–eGFP–MBP–His proteins, BL21-DE3 Escherichia coli cells were cultured at 37 °C to an OD at 600 nm of 0.6–0.9, and induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 18 °C for 16 h. The cells were subsequently collected, pelleted and resuspended in a binding buffer containing 20 mM Tris-HCl (pH 8.0), 1 M NaCl, 10% (v/v) glycerol, 1 mM DTT, and EDTA-free protease inhibitor cocktail according to the manufacturer’s instructions. The cells were lysed by sonication and centrifuged at 10,000g for 20 min. The proteins were purified over pre-packed Dextrin Sepharose High Performance MBPTrap HP (MBPTrap HP columns, GE) and eluted by using the binding buffer containing 10 mM maltose. Proteins were further purified over pre-packed Ni Sepharose High Performance HisTrap HP (GE) and eluted with a buffer containing 50 mM Tris (pH 7.4), 500 mM NaCl and 400 mM imidazole. Protein concentration was determined by Bradford assay (Bio-Rad). The eluted fractions were analysed by using denaturing SDS–PAGE, and the purities of the recombinant proteins were assessed by SDS–PAGE analysis.