P 10 desalting column

DoPhPC and DoPhPE in a proportion of 9:1 were dissolved in a chloroform:methanol (2:1) solution. Squalane was dissolved in chloroform and added at different proportions to obtain 1 mol%, 2.5 mol%, 5 mol% and 10 mol%, respectively. Such solutions were vortexed, dried under a steam of nitrogen gas and left overnight under high vacuum to complete evaporation. Then, the lipid film was rehydrated with a given buffer, followed by vortexing, sonication for five minutes and five cycles of freezing/thawing. To form large unilamellar vesicles (LUVs), the solution was passed through a 100 nm polycarbonate filter by pressure extrusion 11 times at 55 °C using a Mini Extruder^® from Avanti Polar Lipids Inc. (Albaster, AL, USA). Consecutively, it was cooled down and the free dye was removed by chromatography over a prepacked P-10 desalting column from GE Healthcare. Liposomes were kept in ice and used immediately.

Site-specific labeling of αS was performed in an αS variant with a single engineered cysteine at position 122 (αS N122C). This variant was expressed and purified as described above but including 5 mM DTT in all purification steps. The protein was labeled with maleimide-modified Alexa Fluor 488 (AF488) (Invitrogen, Carlsbad, USA) for 15–20 h at 4 °C in the dark. After quenching the reaction with 10 mM DTT, free unreacted dye in the protein solution was subsequently separated using a P10 desalting column (GE Healthcare, Waukesha, USA), and the labeled protein solution was flash frozen with liquid nitrogen and stored at −80 °C. The different peptides, PSMα3, dPSMα3, and LL-37, were labeled at a single engineered cysteine at the N-terminus with maleimide-modified Atto647N (ATTO-TEC, Siegen, Germany). The same labeling and purification strategy were followed as for αS, although in this case the unreacted free dye was removed from the protein solution using a polyacrylamide desalting column (Thermo Fisher Scientific, Waltham, USA). Two cleaning steps were required to remove completely the free dye from the labeled peptide solution.