Figure S3. SDS–PAGE gels documenting expression and purification of D. melanogaster cytoplasmic domain of atlastin (His6-cytATL) and the dimerization mutant His6-cytATL R192Q.

(A) First His6-cytATL protein purification batch using Ni sepharose beads (see the Materials and Methods section for detailed protocol). 1 – Marker (NZY tech protein marker III). 2 – Supernatant after lysis. 3 – Supernatant after incubation with beads. 4, 5, 6, and 7 are fraction eluted with 80, 150, 300, and 400 mM imidazole, respectively. Pooled fractions (6 and 7) were dialyzed overnight into storage buffer, concentrated to 60 mg/ml, flash frozen in liquid nitrogen. Theoretical His₆-cytATL protein size is 48 kDa. (A, B) Second purification batch using the same protocol as in (A) but with increased volume of beads up to 3 ml. Rows: 1 – Marker, 2 – supernatant after lysis, 3 – supernatant after incubation with beads. 4, 5, 6, 7, and 8 – fraction eluted with 40, 80, 150, 300, and 400 mM imidazole, respectively. 9 – Marker. (C) Fractions 6 (lane 3 and 4 with 5 and 10 μl purified protein, respectively) and 8 (lane 5 with 5 μl purified protein) shown in B. (D) His6-cytATL R192Q protein purification batch using Talon resin (see the Materials and Methods section for detailed protocol). 1 – Marker (NZY tech protein marker III). 2 – Supernatant after lysis. 3 – Supernatant after incubation with beads. 4–6 – washes, 7–9 – fractions eluted with 20, 80, and 150 mM imidazole, respectively. Fraction 9 was buffer exchanged into storage buffer, concentrated to 36 mg/ml and immediately flash frozen in liquid nitrogen. Theoretical His₆-cytATL R192Q protein size is 48 kDa.