Minive system

Denaturing one-dimensional electrophoresis was performed using sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis with a 12.5% concentration of acrylamide (Sigma Aldrich, USA) in the separating gel and 5% concentration in the stacking gel [25 (link),26 (link)]. Combs with a 5 mm tooth width were used to form pockets for the samples. In order to reduce disulfide bonds, the analyzed samples were placed in the sample buffer (2% lithium dodecyl sulfate (LDS), 0.065 M Tris-HCl (pH 6.8), 1% dithiothreitol (DTT), 10% glycerol, and 0.01% bromophenol blue (all from Sigma Aldrich, USA)) and were heated in a boiling water bath for 2 min. An amount of 1 μL of the Precision Plus Protein Standard (BioRad, Hercules, CA, USA) was loaded as a marker. A maximum of 40 μL (32 μL of the analyzed sample and 8 μL of the buffer) was applied to the lane. Electrophoretic separation (SDS-PAGE) of the sample proteins was performed using the Hoefer miniVE system (Hoefer Inc., Holliston, MA, USA) (gel size of 80 × 90 × 1 mm). Gels were stained with Coomassie Blue R-350 (GE Healthcare, USA) for 2 h.

The lyophilized venom was diluted in tridistillated water and the protein concentration was determined [28 (link)] using as protein standard BSA (Sigma-Aldrich, catalog number 05470, St. Louis, MO, USA) at different concentrations. SDS-PAGE using 20 µg of venom protein was analyzed under reducing (4% of β-mercaptoethanol and 5 min at boiling water temperature) and non-reducing conditions [29 (link)], using a 10% or 12% polyacrylamide gel in a Mini Protean II unit (Bio-Rad Hercules, CA, USA) or a Hoefer MiniVE system (Hoefer, Holliston, MA, USA). The protein bands were stained with Coomassie Blue G250 dye (Bio-Rad, catalog number 1610406, CA, USA), unstained with a 40% and 10% methanol–acetic acid solution, respectively, and imaged on an HP Scanjet 4570c scanner.

Twenty microgram of lyophilized venom protein was loaded into a 10 or 12% SDS-PAGE gel and electrophoresed in a Mini Protean II unit (Bio-Rad Hercules, CA, USA) or Hoefer MiniVE system (Hoefer, Holliston, MA, USA) under reducing (4% of β-mercaptoethanol and 5 min at boiling water temperature) and non-reducing conditions [62 (link)]. Seven microliter of unstained (Thermo Fisher Scientific, Waltham, MA, USA) or pre-stained (Sigma-Aldrich) MWM standards was used for molecular weight estimation. The gel was stained with 0.1% Coomassie brilliant blue R-250 (Bio-Rad) in 40% methanol and 10% acetic acid (v/v) overnight, de-stained in 40% methanol and 10% glacial acetic acid (v/v), and imaged on an HP Scanjet 4570c scanner.