Native page 4 16 bis tris protein gels

Crude membranes from High Five insect cells (Thermo Fisher Scientific, Waltham, MA) expressing wild-type (WT)–hP-gp, or its double mutant EQ–hP-gp (E556Q/E1201Q), with a 6-histidine tag at the C-terminal end were isolated as described previously (Kerr et al., 2001 (link)). The membranes (150–250 mg of protein) were solubilized by using 2% n-dodecyl β-d-maltoside in a buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 15% glycerol, 5 mM β-mercaptoethanol, 20 mM imidazole, and UltraCruz EDTA-free protease inhibitor cocktail tablets (Santa Cruz Biotechnology, Dallas, TX). The solubilized extract was centrifuged at 38,000 rpm (Ti-45 rotor; Beckman Coulter, Brea, CA) for 45 minutes at 4°C. The supernatant was incubated with Ni-NTA resin (Qiagen Inc., Valencia, CA) pre-equilibrated in solubilization buffer with 0.09% n-dodecyl β-d-maltoside for 14–16 hours at 4°C. The beads were washed and P-gp was eluted with the same buffer containing 300 mM imidazole. Fab isolated from UIC2 monoclonal antibody was then added at a molar ratio of P-gp:Fab (1:3) and incubated at 4°C for 15 minutes. The hP-gp–Fab complex was centrifuged at 300,000 × g for 45 minutes. The complex formation was evaluated on native PAGE 4–16% Bis-Tris protein gels (Thermo Fisher Scientific, Waltham, MA).