N dodecyl β d maltopyranoside ddm

All subsequent steps were performed at 4°C. Membranes at 3 mg/ml protein concentration were solubilized using 1% (wt/vol) n-dodecyl-β-D-maltopyranoside (DDM; Affymetrix) for 1 h in purification buffer (20 mM Tris-Base, 150 mM NaCl, pH 7.4, 0.05% DDM, and 10% glycerol). After ultracentrifugation (2 h, 200,000×g, 4°C), the soluble fraction was incubated for 3 h at 4°C with equilibrated Ni²⁺–nitrilotriacetic acid (NTA) Superflow beads (QIAGEN) with washing buffer (20 mM Tris-Base, 150 mM NaCl, pH 7.4, 0.05% DDM, 10% glycerol, and 20 mM imidazole). Protein-bound beads were washed three times with 10 column volumes of washing buffer before elution with 15 ml of elution buffer (washing buffer supplemented with 350 mM imidazole). The purified protein was concentrated by centrifugation in an Amicon Ultra (100,000 D molecular weight cut-off; Millipore) at 3,220xg until reaching the desired protein concentration. To assess BasC quality and purity after Ni²⁺-NTA purification, size exclusion chromatography (SEC) of GFP-fused BasC was performed in a Superdex 200 10/300 GL (GE Healthcare) in SEC buffer (20 mM Tris-Base, 150 mM NaCl, pH 7.4, and 0.05% DDM). AdiC was purified for functional studies as previously described (Kowalczyk et al., 2011 (link)).