Anti atp5a1 antibody

The biotin switch assay was carried out as described previously with some modifications [16 ]. Cells or liver tissue was homogenized in HEN buffer (250 mM Hepes-NaOH pH 7.7 supplemented with 1 mM EDTA, 0.1 mM neocuproine) containing 150 μM deferoxamine, 1% Nonidet-P40 (NP-40), and protease/phosphatase inhibitors. Homogenized samples were sonicated and centrifuged at 16,000 g for 15 min at 4°C. The supernatant samples were placed into 10K molecular weight concentrator and the retentates were used for protein determination. Lysates were diluted to reach 5.5 mg/ml final protein concentration and were added to HEN buffer supplemented with 2.5% SDS and 20 mM methyl methanethiosulfonate (MMTS). The samples were frequently shaked at 50°C for 25 min. Then, the MMTS was removed by adding acetone and in the meanwhile, the proteins were also precipitated at −20°C for 45 min. Pellets were resuspended in HEN buffer containing 1 % SDS and 4 mM biotin-N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio) propinamide (HPDP). After 3-hour-long incubation at 25°C, biotinylated proteins were purified by streptavidin-agarose beads. The biotinylated proteins were eluted in 2× Laemmeli sample buffer and subjected to Western blotting with anti-ATP5A1 antibody (Abcam, Cambridge, MA, USA), anti-GAPDH antibody (Proteintech Group, Inc., Rosemont, IL) or anti-tetra His antibody (Qiagen, Hilden, Germany).