8 anilino 1 naphthalenesulfonic acid

Dextran (MW: 60,000–90,000) was purchased from Chanshou Biological Co., Ltd. (Jiashu Province, China). SPI was obtained from Wonderful Tech. Co. (Shandong Province, China), containing (on dry basis) 6.5% moisture, 1.0% ash, 0.2% lipid, and 90.2% protein (determined by Kjeldahl method, N×6.25). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), o-phthaldialdehyde (OPA), and 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acrylamide (99%) was purchased from Sigma (Deisenhofen, Germany) and HMF (98%) was from Acros (Geel, Belgium). All other chemicals used were of analytical grade and procured from Merck (Darmstadt, Germany).

To detect hydrophobic molecules, TLC plates were sprayed with ANS reagent (8-Anilino-1-naphthalenesulfonic acid, Sigma) at 0.2% in methanol (Zbierzak et al., 2011 ) and lipids were visualized as fluorescent spots under UV light (366 nm). Photographs of TLC plates were processed with ImageJ (Schneider et al., 2012 (link)) as follows: contrast was increased; images were converted to black and white and inverted for easier visualization. In order to obtain a rough quantification of the lipids, ImageJ was used to measure the integrated density of each spot from the ANS-stained TLC plates. Spots corresponding to neutral lipids were not included in the quantification analysis.
In some experiments, 2D-TLC plates were stained with ninhydrin (Sigma), Phospray reagent (Supelco), or orcinol (Sigma) for detection of aminolipids, phospholipids, and glycolipids, respectively. Identity of the different lipids was assigned by comparison with R_f from lipid standards analyzed in the same TLC systems (Supplementary Figure S1), coupled with the use of different TLC stainings. Identification of some molecules was also complemented with MS analyses.

The critical micelle concentration (cmc) was measured by fluorimetric titration of ANS (8-Anilino-1-naphthalenesulfonic acid, obtained from Sigma-Adrich) with different detergents as described^{63 (link)}. Briefly, titration was carried out at 4 °C by adding increasing volumes of 1 µl to 20 µl of a detergent stock solution (200 mM FC12, 20 mM FC14, 2 mM FC16, 25 mM DDM) to 50 µM ANS in either SEC or CD buffer (2.8 ml). The emission of ANS was monitored at 490 nm with 370 nm excitation (bandwidth 2 nm). The fluorescence intensity was corrected for dilution. The cmc was obtained from the intersection point of two linear fits describing the curves (see Supplementary Figure S11b).

Protein surface hydrophobicity of the samples was measured according to the modified method described by Mishyna et al. (2019) [22 (link)]. Each sample was dissolved in 0.2 M phosphate buffer (pH 7.0) and stirred for 1 h. After centrifugation at 3200× g for 20 min, the protein concentration in the supernatant was measured by the Bradford method and adjusted to various concentrations (0.005%, 0.01%, and 0.02% w/v). Fifteen microliters of 8-anilino-1-naphthalenesulfonic acid (0.008 M; Sigma-Aldrich) were added to 3 mL aliquots of each supernatant and left for 15 min in the dark. The fluorescence intensity of the soluble protein was measured by a fluorescence spectrophotometer (LS-55, Perkin Elmer, Waltham, MA, USA) at an excitation wavelength of 390 nm and emission wavelength of 480 nm. The protein surface hydrophobicity was estimated as the slope of the plot of protein concentration vs. fluorescence intensity.

Thioflavin T (ThT), tris(hydroxymethyl)aminomethane, phenylmethylsulfonyl fluoride (PMSF), Congo red, 8-Anilino-1-naphthalenesulfonic acid (ANS), and 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) were purchased from Sigma (St.Louis, MO, USA). KCl, NaCl, ZnCl₂, CuCl₂, CaCl₂, MgCl₂, ethylenediaminetetraacetic acid (EDTA), ammonium sulfate, NaH₂PO₄, Na₂HPO₄, and KH₂PO₄ were obtained from Merck. pT7-7 asyn WT plasmid containing α-Syn gene was obtained from addgene. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) was purchased from CinnaGen. The HiTrap Q FF anion exchange chromatography column was from GE Healthcare. Dulbecco's Modified Eagles Medium (DMEM), fetal bovine serum (FBS), and trypsin were all from Gibco. Penicillin–streptomycin solution was purchased from BIO-IDEA. The cell culture flasks and plates were obtained from SPL.