Coomassie g 250

Spartin proteins including HsSpartin, CeSpartin and CtSpartinL were expressed in Expi293 cells, and purified using buffer A (50 mM HEPES, 500 mM Nacl, pH8.0, 1 mM TCEP, 10% glycerol) using anti-FLAG resin. Proteins were quantified using BSA standards in SDS-PAGE. Proteins (1–2 μg) together with BSA (1 μg) were loaded onto native PAGE 4%−16% Bis-Tris gel (BN1002BOX, invitrogen) for electrophoresis at 4C using NativePAGE running buffer and NativePAGE Cathode buffer (contains 0.02% Coomassie G-250, BN2007, invitrogen). NativeMark unstained protein standards (LC0725, invitrogen) and NativePAGE sample buffer (BN2003, invitrogen) were used. Protein gels were fixed and destained according to manufacturer direction. Distinct from native gels in the lipid-co-migration assay (above), where samples migrate according to their charge/mass ratio, here the Coomassie dye in the cathode buffer coats protein sample, which consequently migrates according to MW (28 (link)).

Enriched biotinylated proteins by magnetic streptavidin beads were separated on 10% SDS-PAGE gels and stained with Coomassie G-250 (Invitrogen). Lanes for each replicate were manually cut into 3–4 gel bands. Each band was destained with 40% acetonitrile/50 mM NH₄HCO₃. The gels were dehydrated with 100% acetonitrile and dried for 5 min using a SpeedVac. Disulfide bonds were reduced with DTT (10 mM, 56 °C, 45 min), and the free sulfhydryl groups were alkylated with iodoacetamide (55 mM, 25 °C, 60 min in the dark). Gel pieces were sequentially washed with 50 mM NH₄HCO₃, 50% acetonitrile/50 mM NH₄HCO₃, and dehydrated with 100% acetonitrile. After drying with a SpeedVac, the gel was rehydrated using 100 ng/μL trypsin (Promega, V5113) on ice for 30 min, and the digestion was carried out at 37 °C overnight and quenched with 1.0% TFA. The tryptic peptides were extracted twice with 60% acetonitrile containing 0.1% TFA, and the combined digest solution was dried using a SpeedVac.

Mitochondria were resuspended in solubilization buffer (2% digitonin/DDM = 2 mg digitonin/DDM per mg protein) and incubated on ice for 10 min. The samples were centrifuged (7 min, 25 000 g), the supernatant was saved and mixed with 0.5% sample additive (NativePAGE^TM 5% G-250 Sample Additive; Thermo Fisher Scientific). The samples were loaded (100 μg per sample) on a 3–12% NativePage Bis-Tris gel (Thermo Fisher Scientific) using NativePAGE^TM 1X Running Buffer as anode buffer and NativePAGE^TM 1X Running Buffer supplemented with 0.02% Coomassie G-250 as cathode buffer (Thermo Fisher Scientific). The gel was run until 1/3 of the gel at 150 mV, then the cathode buffer was exchanged to NativePAGE^TM 1X Running Buffer and the gel was run until the end at 250 mV. Subsequently, the gel was either transferred to a PVDF membrane (Bio-Rad; 100° mA, 90° min) or a second dimension denaturing polyacrylamide gel was run. For the second dimension each sample lane was cut out of the gel entirely or partly, incubated 20 min in running buffer containing 100 mM DTT and inserted in a 90° angle in a denaturing polyacrylamide gel (16% acrylamide, 0.2% bisacrylamide; 30 mA, 50 min). The proteins were subsequently transferred to a nitrocellulose membrane (Amersham^TM Protran^® Premium Western blotting membranes). Antibodies were prepared in 5% milk dissolved in TBS.