Fluorescence-based Peptide Transport Assay

Transport assays were carried out as described previously (22 (link)), employing a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) to measure the change in fluorescence of the pH-sensitive dye pyranine. Dual fluorescence excitation was set to 460/415 nm with emission at 510 nm. Transport was initiated following addition of 1 μM valinomycin. Competition assays were performed at 30 °C, with samples taken at specified time points. Proteoliposomes were immediately filtered onto a 0.22-μm cellulose filter (Merck Millipore) using a vacuum manifold and washed twice with 2 mL cold H₂O. The amount of peptide transported into the liposomes was calculated based on the specific activity for each peptide. Experiments were performed a minimal of four times to generate an overall mean and SD and the resulting data were analyzed using Prism 7.0 (GraphPad Software).