Ammonium sulfate

The internal dialyzed fraction, containing molecules larger than 3.5 kDa, was subjected to precipitation by ammonium sulfate (Sigma-Aldrich). This fraction was quantified by Bradford (Bio-Rad Laboratories, Hercules, CA, USA) [57 (link)] and diluted in water in a ratio of 1 mg/mL. After this procedure, the material was precipitated by saturation in 0–30%, 30–60%, and 60–90% ammonium sulfate, according to the table available in [58 ]. At each stage of saturation, the material was maintained at 4 °C for 4 h, and subsequently centrifuged at 7500 g for 15 min. The precipitate was diluted in distilled H₂O and dialyzed against a membrane of 3.5 kDa pore size for desalination. The precipitated fractions and the non-precipitated fraction from 60–90% were subjected to biological evaluation of anti-inflammatory action by neutrophil migration.

The 1D gel method utilised the following specific conditions: 1 mm 12% SDS-Page gels with resolving gel layer (3.3 mL Milli-Q H2O, 4 mL 30% acrylamide mix (Bio-Rad, Hercules, CA, USA), 2.5 mL 1.5MTris-HClbuffer (Tris—Sigma-Aldrich, St. Louis, MO, USA; HCl—Univar, Wilnecote, UK), pH 8.8, 100 μL 10% SDS (Sigma-Aldrich, St. Louis, MO, USA), 4 μL TEMED (Bio-Rad, Hercules, CA, USA), 100 μL 10% APS (Bio-Rad, Hercules, CA, USA); 20 μg venom sample per lane after dissolving in 3 μL of 4× sample loading buffer (Bio-Rad, Hercules, CA, USA) brought up to 12 μL total volume, with DTT (Sigma-Aldrich, St. Louis, MO, USA); reducing conditions were 3 min incubation at 100 °C; gels were run at room temperature for at 120 V (Mini Protean3 power-pack from Bio-Rad, Hercules, CA, USA) for 20 min and then 140 V for 60 min; runs were stopped when dye front was less than 10 mm from the base of the gel. Gels were stained with colloidal coomassie brilliant blue G250 (34% methanol (VWR Chemicals, Tingalpa, QLD, Australia), 3% orthophosphoric acid (Merck, Darmstadt, Germany) 170 g/L ammonium sulfate (Bio-Rad, Hercules, CA, USA), 1 g/L coomassie blue G250 (Bio-Rad, Hercules, CA, USA)) overnight and then destained in 1% acetic acid (Univar, Wilnecote, UK).