Talon chromatography

For biochemistry assays (Fig. 5A-G), pET28c plasmid constructs were generated for NMNAT2^R232Q and NMNAT2^Q135Pfs*44 pET28c to produce recombinant proteins with an N-terminal His tag and linker (MGSSHHHHHHSSGLVPRGSH) for affinity purification that matched a previously generated NMNAT2^WT construct ^{33 (link)}. Expression was carried out in E. coli BL21(D3) cells (Invitrogen) following 0.5 mM IPTG induction for 4 h at 25°C with subsequent purification using TALON chromatography (Clontech) as described ³⁴ . The purified proteins were desalted on PD-10 columns (GE Healthcare) in 50 mM HEPES/NaOH buffer, pH 7.5, 1 mM Tris(2-carboxyethyl)phosphine (TCEP), 20 % glycerol, and stored at −80 °C. Their amount was measured by the Bio-Rad protein assay. Their purity was evaluated on SDS polyacrylamide gels either after Coomassie staining or immunoblotting. Proteins were transferred from gels to Immobilon-P membrane (Millipore) and probed with antibodies as described ^{20 (link)}. Monoclonal anti-NMNAT2 (1:1,000 Abcam AB5698) or anti-tetra His (0.1 μg/ml Qiagen 34670) were used as primary antibodies, followed by appropriate HRP-conjugated secondary antibodies. SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used for detection on an Alliance chemiluminescence imaging system (UVITEC Cambridge).