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Figure 1
Isolation of a Drosophila TOM complex. (a) Schematic representation of the UAS–GAL4 protein-expression system in Drosophila. The promoter (GMR) drives ectopic expression of Tom40 in fly retina via the transcription factor GAL4 and the GAL4 response element UAS. This image was created with BioRender.com. (b) Workflow of protein purification. Fly heads were separated using a metal sieve stack. Fly heads collected in the 500 µm compartment were homogenized and membranes were prepared by ultracentrifugation of the lysate. Membranes were detergent-solubilized and the protein was affinity-purified using anti-FLAG resin. (c) Comparative BN-PAGE Western blot (anti-HA) analysis of eye membranes, expressing Tom40 (co-expressed with Myc), solubilized with either digitonin or n-tetradecyl-β-D-maltopyranoside (TetraDM)–cholesterol hemisuccinate (CHS). Additional bands migrating close to the 146 and 720 kDa markers indicate dissociated complexes and super-complexes, respectively. (d) Silver-stained SDS–PAGE gel analysis of immuno-affinity-purified TOM complex reconstituted into non-ionic amphipol. Protein identities of prominent gel bands detected by tryptic digest mass spectrometry are labeled with asterisks.

IUCrJ
Volume 12| Part 1| January 2025| Pages 49-61
ISSN: 2052-2525
https://doi.org/10.1107/S2052252524011011