Max xp benchtop centrifuge

POPC and gangliosides were purchased from Avanti Polar Lipids (Alabaster, AL). POPC or POPC mixed with 1% ganglioside mixture was dried with nitrogen gas and further dried overnight at room temperature under vacuum to form the lipid films. The lipids were then resuspended with Tris buffer. The resuspended lipids were extruded through a polycarbonate filter (Whatman, 200-nm pore size) by 20 strokes to form liposomes. Liposomes (75 μl) were incubated with H_C/B proteins (1 μM) at room temperature for 30 minutes. The liposome–protein mixture was then added to sucrose solution to reach a final sucrose concentration of 30%, which served as the bottom layer in the centrifuge tube. The 200 μl of 25% sucrose was loaded on the top of the bottom layer, followed by 50 μl of Tris buffer. Loaded samples with sucrose gradient were centrifuged at 240,000g for 1 hour (Beckman TLS-55 rotor, OptiMax MAX-XP benchtop centrifuge). After centrifugation, 50-μl solutions on the top of the gradient were collected and subjected to immunoblot analysis.

PC was dissolved in chloroform. Gangliosides were dissolved in chloroform:methanol (3:1). PC alone or PC mixed with gangliosides (1%) was dried under nitrogen gas. Lipid films were re-hydrated with the lipid reconstitution buffer (30 mM Tris, 150 mM NaCl, 2 mM MgCl₂, 2 mM DTT, pH 7.5). Re-suspended lipids were mixed using a shaker at RT for 1 h. Liposomes were generated from re-suspended lipids with an extruder (200 nm pore size, 20 strokes manually, Avanti). Liposomes (75 µl) were incubated with 1 µM proteins (BoNT/DC-H_C WT or mutants) in a total volume of 150 µl for 30 min at RT. The liposome–protein mixtures were then added to 100 µl 75% sucrose solution (in lipid reconstitution buffer) to get 250 µl 30% sucrose solution that were loaded as the bottom layer in the centrifuge tube, followed by 200 µl 25% sucrose, and 50 µl lipid reconstitution buffer, as depicted in Fig. 5a. Loaded sucrose gradients were centrifuged at 240,000 × g for 1 h (Beckman TLS-55 rotor, OptiMax MAX-XP benchtop centrifuge). After the centrifugation, 50 µl solutions were taken from the top of the centrifuge tube and subjected to immunoblot analysis.