3h succinic acid

Proteoliposomes were prepared for transport assays by extruding them through a 400 nm filter 11 times and concentrating them to 80 mg/ml lipid using ultracentrifugation. The transport assays were started by adding the proteoliposomes to Reaction Buffer (20 mM Tris/HEPES, pH 7.5, 100 mM NaCl, 100 mM KCl, 1 μM valinomycin, 1 μM [³H]-succinic acid (American Radiolabeled Chemicals). At the indicated times, samples were taken and the reaction was terminated by adding the sample to ice cold Quench buffer (20 mM Tris/HEPES, pH 7.5, 200 mM ChCl) and rapidly filtering the sample through a 200 nm nitrocellulose filter (Millipore). The filter was washed with 3 ml Quench buffer, the filters were dissolved in FilterCount liquid scintillation cocktail (PerkinElmer) and the [³H]-succinic acid internalized by the proteoliposomes was counted using a Trilux beta counter (PerkinElmer).