Lipid Strips

Lipid strips were purchased from Echelon. The strips were washed with 4 × 5 mL of wash buffer [10 mM phosphate buffer (pH 7.4), 2.7 mM potassium chloride, 137 mM sodium chloride, 0.05% Tween 20, and 0.5% BSA] and blocked with 5 mL of blocking buffer [10 mM phosphate buffer (pH 7.4), 2.7 mM potassium chloride, 137 mM sodium chloride, 0.05% Tween 20, and 1% BSA] for 1 h at room temperature. The strips were washed, 4 × 5 mL for 5 min, and thereafter incubated with a 6 μM protein solution (FadD13 in wash buffer) for 1 h at room temperature. The strips were subsequently washed, 4 × 5 mL for 5 min, and incubated with the primary antibody (mouse anti-His) diluted 1:2000 in wash buffer for 1 h at room temperature. The strips were washed again, 4 × 5 mL for 5 min, and incubated with a secondary antibody (donkey anti-mouse) and diluted 1:2000 for 1 h at room temperature. Afterward, the strips were washed, 4 × 5 mL for 5 min, and detected with 2 mL of a TMB solution.