Ni sepharose high performance his trap hp

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was purchased from Avanti Polar Lipids (USA), C12E8 (Octaethylene glycol monododecyl ether, ≥ 98% purity, ref. P9825), Triton X-100 (BioXtra, ref. 9284), ATP (Adenosine 5′-triphosphate disodium salt hydrate, ref. A2383), ADP (Adenosine 5′-diphosphate ≥95%, ref. 01905), L-Lactic Dehydrogenase from rabbit muscle (ref. L1254), NADH (β-Nicotinamide adenine dinucleotide, reduced disodium salt, ref. N0786), Pyruvate Kinase from rabbit muscle (ref. P7768), PEP (Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt, ref. P7252), roxithromycin (ref. R4394) and QDs (CdTe core-type, ref. 777935) were purchased from Sigma, SM2 Bio-beads were obtained from Bio-Rad. Ni Sepharose High Performance (His Trap HP) and Superose 6 10/300 column were purchased from GE Healthcare.

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.