The following synthetic lipids were used: POPG, POPE and POPC (Avanti Polar lipids). Additionally a bovine brain extract, Folch Fraction I (Sigma Aldrich) was used. For tubulation experiments large uni-lamellar vesicles (LUVs) or multi-lamellar vesicles (MLVs) were prepared. The dried lipid film, containing 2POPG:1POPE (w/w) was hydrated with buffer (20 mM Hepes pH 7.4) to achieve MLVs. LUVs were obtained by extrusion with a filter membrane (200 nm) (Avanti Polar lipids). The lipid condition was determined based on the screenings done by Isas et al.13 (link). All prepared lipids were either immediately used or stored at 4 °C for 2–3 days.
For the tubulation kinetics experiments various amphiphysin/BIN1 fragments (20 μM, 15 μM or 6 uM of N-BAR and N-BAR-deltaH0 or 6 μM of FL and deltaH0) were incubated with 180 μM of liposomes. The tubulation was measured by absorbance spectrometry at 400 nm and by negative-stain EM. For tubulation assays the absorbance was followed for 120 min. Additionally, aliquots of different proteins were taken at several time points up to 45 min and samples were assessed with negative-stain EM.
For the tubulation kinetics experiments various amphiphysin/BIN1 fragments (20 μM, 15 μM or 6 uM of N-BAR and N-BAR-deltaH0 or 6 μM of FL and deltaH0) were incubated with 180 μM of liposomes. The tubulation was measured by absorbance spectrometry at 400 nm and by negative-stain EM. For tubulation assays the absorbance was followed for 120 min. Additionally, aliquots of different proteins were taken at several time points up to 45 min and samples were assessed with negative-stain EM.
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