Sm2 bio beads

Lipid mixtures, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-snglycero-3-phospho-(1'-rac-glycerol) (POPG) at a 3:1 ratio (w/w) or POPE, POPG and PI(4,5)P2 at a 3:1:0.1 ratio were dried under an argon stream, and placed in a vacuum chamber overnight. Dried lipids were suspended by sonication in buffer containing 20 mM Tris pH 8 and 160 mM KCl. DDM was added and mixed with the lipid suspension for 1 hour at room temperature. The final DDM concentration was 10 mM and the lipid concentration 10 mg/ml. Purified channel complex was added to the lipid/DDM mixture at a protein-to-lipid ratio of 1:250 (w/w). After 30-min incubation at room temperature, SM2 biobeads (Bio-Rad) were added to remove DDM by incubating the mixture at 4 °C overni ght.
Proteoliposome vesicles were collected, briefly sonicated and diluted 25 times in buffer containing 20 mM Tris pH 8 and 160 mM NaCl and prior to the assay. 6 μl vesicles was mixed with 6 μl ACMA solution (7.5 μM) and 12 μl buffer (20 mM Tris pH 8 and 160 mM NaCl). Fluorescence was recorded every 5 seconds (λE_X = 410 nm, λE_M = 490 nm) using a plate reader (Tecan Infinite M1000). After the ACMA fluorescence stabilized, K⁺ flux was initiated by addition of 6 μl CCCP (40 μM). At the end of the assay, 2 μM valinomycin was added to release K⁺ from all vesicles and yield a minimum baseline fluorescence.

A POPC:POPG lipid bilayer was used to mimic phospholipids typically found in mammalian membranes^{[9 (link), 12 (link), 15 (link)–17 (link), 25 (link), 26 (link)]}. POPC and POPG powdered lipids (Avanti, Alabaster, AL) were dissolved to a total final concentration of 5 mM (9/1 POPC/POPG molar ratio) in 5 mM HEPES and 0.5% DM (n-Dodecyl-β-D-maltoside) (pH 7.0) by repeating at least 10 freeze/thaw cycles until the solutions were clear^{[12 (link)]}. SM2 Bio Beads (Bio-Rad) were washed with 8× 20 mL volumes of methanol followed by 8× 20 mL volumes of water prior to use. Samples were reconstituted by mixing 1 mL of the lipid slurry with 157 μg of KCNE1 protein in 0.2% SDS (500/1 lipid/protein molar ratio) and repeating 2 freeze/thaw cycles. The lipid/protein mixture was then added to 300 mg of wet Bio Beads and nutated at room temperature for 2 hours. Previous studies showed that this amount of Bio Beads effectively removes all detergent and forms MLVs^{[27 (link)]}. The resulting sample was centrifuged at 2,000 g for 5 minutes to remove excess Bio Beads. The supernatant was concentrated by ultracentrifugation at 300,000 g for 30 minutes. The proteoliposome pellet was thoroughly resuspended in 50 μL of 100 mM NaH₂PO₄ (pH 7.2) and immediately used in spectroscopic studies.