Quantifying Cell Surface K10.1-BBS Expression

Cells expressing K_V10.1-BBS (52 (link)) were incubated for 10 min on ice with 2.5 μg/ml α-bungarotoxin (BTX)-biotin (Invitrogen), washed twice with ice-cold PBS, scraped, and centrifuged at 800 × g for 3 min. Cell pellets were resuspended in 20 mm Tris-HCl, 150 mm NaCl, 5 mm MgCl₂, and 1% Nonidet P-40, pH 7.4, containing protease inhibitors. The lysates were passed several times through a 25-gauge needle, placed in microcentrifuge tubes, incubated for 20 min on ice, and finally centrifuged at 16,100 × g for 15 min at 4 °C. Streptavidin-coated plates (Thermo Scientific) were washed twice with PBS containing 0.05% Tween 20, 0.1% BSA, and 0.1% Triton X-100. 30 and 150 μg of protein (in triplicates) were then incubated for 30 min on ice. The plates were washed twice and then blocked for 30 min with NPE (150 mm NaCl, 5 mm EDTA, 50 mm Tris, 5 mm KCl, and 1% Nonidet P-40, pH 7.5) containing 1% casein. Anti-K_V10.1 antibody (mAb62) was diluted in NPE with 0.1% casein and used at 2.5 μg/ml for 90 min. The wells were washed three times with NPE with 0.1% casein and blocked again in NPE with 1% casein for 30 min. Secondary antibody (1:500 ECL HRP-linked anti-mouse IgG (GE Healthcare)) was then incubated for 90 min. After seven washes with NPE-0.1% casein, peroxidase activity was determined using ABTS as described above.