4.4. Sedimentation Assay

Prior to each experiment, protein samples were thawed on ice and centrifuged for 10 min at 16,000× g to remove potential protein aggregates. A reaction mixture usually contained 5 µM DivIVA_Cd or 10 µM Δ60-DivIVA_Cd and 1 mM SUVs and was incubated for 15 min at 30 °C. Samples were centrifuged for 10 min at 16,000× g and 30 °C. Even this low centrifugation force was sufficient to sediment the lipid vesicles to which DivIVA_Cd was bound. We also tested higher centrifugation force (100,000× g) and obtained similar results (not shown). The supernatant containing the unbound protein fraction was collected and 4× SDS sample buffer was added to reach a 1× final concentration. Pellets containing the liposome-bound protein fraction were resuspended directly in 1× SDS sample buffer. Samples were analysed using SDS-PAGE and the ratio between bound and unbound protein was quantified using the Gel Analysis Fiji plugin. The results were plotted in GraphPad Prism 8 and evaluated using a two-tailed t-test. P values were not corrected for multiple comparisons and apply individually to each value, not to the entire family of comparisons.