Rotavapor model r 114

Detergent solubilized Pgp was integrated into liposomes as previously described [e.g. [44 (link)]]. Briefly, 80% w/v Avanti extract polar Escherichia (E.) coli lipids (Avanti Polar Lipids, Alabaster, AL) and 20% w/v cholesterol were solubilized in chloroform to a final concentration of 10 mg mL⁻¹. This solution was evaporated in a Buchi Rotavapor Model R-114 (Buchi, Switzerland) until complete dryness was achieved and a lipid film was formed. This lipid film was rehydrated in 50 mM Tris-HCl and 0.1 mM ethylene glycol tetraacetic acid (EGTA), pH 7.4 and underwent 10 freeze thaw cycles in liquid nitrogen. The resuspended lipid film was extruded 11 times through a LIPEX extruder (Northern Lipids, Burnaby, British Columbia, Canada) with a 400 nm cutoff Millipore filter (EMD Millipore Billerica, MA). The purified Pgp product was dialyzed to remove excess detergent against HEPES buffer (20 mM HEPES, 100 mM NaCl, 5mM MgCl₂, 2 mM DTT, pH 7.4). Dialyzed Pgp at 25 μM was passively integrated into 4 mg ml⁻¹ lipososomes to a final concentration of 6.25 μM Pgp mg⁻¹ ml liposomes. These proteoliposomes were dialyzed against HEPES buffer to remove the remaining detergent and stored at −80 °C.