Qproteome plasma membrane protein kit

The ABCG2-associated ATPase activity was measured as previously described (Ji et al., 2018 (link)). Membranes were prepared using protein extraction kit (Qproteome Plasma Membrane Protein Kit, Qiagen) from transfected ABCG2 overexpression HEK293 cells. The membranes were incubated with ATPase buffer (Ji et al., 2019a (link)) at 37°C for 3 min with or without 0.4 mM sodium vanadate. Gradient concentrations of AZ-628, topotecan or M3814 were added and incubated at 37°C for 5 min then 25 mM Mg-ATP solution were added and incubated at 37°C for 20 min. Reactions were terminated by adding 5% SDS. The amount of inorganic phosphate was quantified using a colorimetric method under 880 nm.

Cells cultured in a 225 cm² flask (approximately 30 × 10⁶ cells) were scraped, using a cell scraper in plain PBS, then centrifuged at 1500 rpm for 5 min. Pelleted cells were used for the plasma membrane isolation using the “Qproteome Plasma Membrane Protein kit” (Qiagen), according to the manufacturer’s instructions. The kit utilizes a ligand specific for molecules on the plasma membrane and magnetic beads that bind to the ligand and ensures the specific isolation of plasma membranes. Isolated plasma membranes were solubilized by Tris-membrane buffer with CHAPS (50 mM Tris-HCL 0.1 mM CaCl₂, 1 mM MgCl₂, 5 mM KCl, 10 mM CHAPS pH 7.4) and centrifuged at 27000 rpm, in Beckman XL-90 Ultracentrifuge (Type 70 Ti Rotor), 4 °C for 1 h. The supernatant was collected and its protein concentration was measured using a spectrophotometer at 280 nm (NanoDrop 2000, Thermo Scientific, Cheshire, UK).