Snakeskindialysis tubing 3.5k mwco

The GO synthesis was carried out using the modified Hummers method, following the methodology proposed by Mangadlao et al. with some modifications [58 (link)]. Briefly, 3 g of graphite were mixed for 10 min in a flat bottom flask, and then 3 g of KMnO₄ were added to the mixture. The reaction remained in constant agitation all the time and every 24 h, 3 g of KMnO₄ were added three more times. The reaction was stopped 24 h after the last addition of KMnO₄ by taking a third of the mixture (approximately 134 mL) and mixing it with 300 mL of an ice water/Milli Q water mixture. After 10 min 2 mL of H₂O₂ were added and stirring was continued for 10 min. The mixture was transferred to 50 mL falcon tubes and centrifuged for 10 min at 4000 rpm. The supernatant was discarded, and the sediment was washed twice with Milli Q water followed by centrifugation. The purification was then carried out using 2-propanol until reaching a neutral pH. The solution was dialyzed for three days in isopropanol using SnakeSkin Dialysis Tubing, 3.5K MWCO (Thermo Scientific, Suwanee, GA, US). Finally, two new washes were made with Milli Q water, after which the graphite oxide was frozen at −40 °C for at least 24 h and lyophilized at −51 °C and 0.12 mBar pressure for 24 h in a Freezone freeze dryer 4.5 (Labconco, Kansas City, MO, US).

The preparation of recombinant destabilase was described in detail earlier^{37 (link)}. Briefly, DNA fragments encoding destabilase isoform Ds2 (UniProt ID Q25091) were synthesized from oligonucleotides. The plasmid pcDNA3.4-Dest2 for the expression of the destabilase gene in the human cell line Expi293F was constructed using a pcDNA3.4-TOPO TA Cloning Kit (Invitrogen, USA). The transient human cell line Expi293F producing destabilase was generated using an Expi293F Expression System Kit (Life Technologies, USA). The cells were transfected by ExpiFectamine 293 transfection agent with pcDNA3.4-Dest2 plasmid followed by incubation for 120 h, the enzyme was secreted into the culture medium. The presence of C-terminal 6xHisTag allowed for the concentration and purification of the target protein using metal chelate chromatography on Ni Sepharose High Performance column (GE Healthcare). The yield of pure destabilase was 20 mg per 1 l of the human cell line culture. Additional purification step was performed using CM Sepharose Fast Flow media (GE Healthcare, USA). The solution of purified destabilase was dialyzed (SnakeSkin Dialysis Tubing 3.5 K MWCO, Thermo Fisher Scientific) against 5 mM Na₂HPO₄ at pH 5.0.

L-α-Phosphatidylcholine of Soy (purity >99%) was purchased from Avanti Polar Lipids, Alabama, USA. Sodium Cholate, Indole, Imiquimod and AmphotericinB were from Sigma Aldrich, St. Louis, USA. SnakeSkin® Dialysis Tubing, 3.5 K MWCO was purchased from Thermo Scientific, USA. Methanol and chloroform were from Emsure, Germany. HEPES was from AppliChem, Germany. All other reagents were of analytical grade. For HPLC measurements, methanol and acetonitrile (Anhedra®) were HPLC quality. Absolute ethanol (Sintorgan®) and dietilamine (Anedra®) were FVpharmaceutical grade. For mobile phases, monobasic potassium phosphate and sodium acetate (Ciccarelli®) pharmaceutical grade were used. All experiments were done with Milli-Q water.