Ampholine

CaCl₂, ≥93.0%; Na₂CO₃, ≥99.0%; Histopaque 1.077, 1.119; Krebs–Ringer solution; Folin–Ciocalteu reagent; human serum albumin fraction V (HSA); methylglyoxal; lucigenin; superoxide dismutase (SOD); horseradish peroxidase (HRP); sodium azid (NaN₃); scopoletin; trisodium citrate; genistein; wortmannin; diphenyleneiodonium chloride (DPI); ethylenedinitrilotetraacetatic acid (EDTA); Tris; 3-((3-cholamidopropyl) dimethylammonio)-1-propanesufonate (CHAPS); nonyl phenoxypolyethoxylethanol (NP40); N,N,N′,N′-tetramethyl ethylenediamine (TEMED); ammonium persulfate; glycerol; sodium dodecyl sulfate (SDS); thiourea; acrylamide; and bisacrilamide were all purchased from Sigma-Aldrich (St. Louis, MO, USA); glycine was purchased from ApliChem GmbH (Darmstadt, Germany); gelatin from Fluka (Seelze, Germany) (; dextran T70 from Roth, Germany; USP-grade urea from Amresco (Solon, OH, USA); 2,5-dihydroxybenzoic acid from Bruker Daltonics (Billerica, MA, USA); cyanines from Lumiprob (Hunt Valley, ML, USA); ampholines, DIGE standards, dithiothreitol (DTT) from BioRad (Hercules, CA, USA); and NaH₂PO₄∙2H₂O, propanol-2, CuSO₄·5H₂O, NaCl, Eur Ph grade CaCl₂·2H₂O and sodium citrate were purchased from “Chimmed”(Moscow, Russia).

Protein extract was prepared with the frozen cysticerci, as previously reported [19 (link)], in 2DE buffer (8 M urea, 50 mM DTT, 2% CHAPS, 2% Ampholine^®, pH 3–10) (Bio-Rad, Hercules, CA, USA) and protease inhibitor (Halt™ Protease Inhibitor Cocktail). The protein concentration was measured using the Bradford method [20 (link)]. A total of 100 µg of protein from each sample was applied to IPG strips (GE HealthCare, Chicago, IL, USA) in an immobilized pH gradient from 3 to 10 by isoelectric focusing at 10,000 V over 8 h. Next, the strips were equilibrated with one wash for 10 min with 8 M urea, 0.375 M Tris, pH 8.8, 2% SDS, 20% glycerol, and 2% (w/v) DTT, and another wash for 10 min with 8 M urea, 0.375 M Tris pH 8.8, 2% SDS, 20% glycerol, and 2.5% (w/v) iodoacetamide. The proteins were separated in a second dimension by SDS-PAGE in a buffer of 25 mM Tris, pH 8.3, 250 mM glycine, and 0.01% SDS.

Trichloroacetic acid (TCA)/acetone method was used for total soluble protein extraction [29 (link)]. Frozen leaf material was crushed using liquid nitrogen, powder obtained were collected in 40 mM Tris-Base (pH 7.5), EDTA (2 mM), 2% (w/v) PVP, β-mercaptoethanol 0.07% (v/v), Triton X100 1% (v/v), PMSF (1 mM) and glycerol 10% (v/v), vortexed and left to room temperature for 2 h, then at 4 °C for 15 min centrifuged at 15,000 rpm. In 10% cold TCA the extract was mixed, additionally β-mercaptoethanol 0.07% (v/v) and EDTA (2 mM) was added in the supernatant, finally kept for overnight at -20 °C. The mixtures were centrifuged at 5000 rpm for 5 min, continued by chilled acetone thrice gentle washings. Obtained pellet was vacuum dried and resuspended in solubilization buffer consisting of urea (7 M), thiourea (2 M), CHAPS (4%), DTT (40 mM) and ampholytes (0.2%) (Ampholine, Bio-Rad, USA) at pH 3–10, followed by centrifuge at 6000 rpm for 5 min at room temperature. Total protein content was quantified by standard method using [30 (link)]. Bovine serum albumin (BSA) served as the protein standard.