Non linear ipg strips

CM was analyzed by 2DE according to Chevallet et al. [12 (link)]. Proteins (60 μg) were resolved on 7 cm nonlinear IPG strips, pH range 3–10 (Bio-Rad), using the in-gel rehydration method. This was followed by a reduction (dithioerythritol) and alkylation (iodoacetamide) of IPG strips, while the second dimensional analysis was performed on 11% SDS-PAGE. Staining of 2DE gels was performed with Coomassie Colloidal Blue. Four biological replicates were analyzed for each cell line.

We performed two dimensional (2D) SDS-PAGE gel electrophoresis experiments using each of the four A. contortrix venoms to compare venom compositional profiles. For each gel, 0.5 mg of venom was prepared for 2D gel electrophoresis using the ReadyPrep™ 2-D Cleanup Kit for isoelectric focusing (IEF) (Bio-Rad) as per the manufacturer’s instructions. Cleaned-up venom samples were then applied to 7 cm, pH 3–10, non-linear IPG strips (Bio-Rad) using the ReadyPrep™ 2-D starter kit (BioRad), as per manufacturer’s instructions, and re-hydrated overnight at room temperature. After re-hydration, IEF was performed using a PROTEAN^® IEF Cell (Bio-Rad) with the manufacturer’s standard electrophoresis protocol for 7 cm IPG strips (default cell temperature = 20 °C; maximum current 50 Ua/strip; voltage = 250 V with linear ramp for 20 min; 4000 V with linear ramp for 2 hours; 4000 V with rapid ramp for 10,000 V-hr). After IEF, IPG strips were equilibrated (as per the ReadyPrep™ 2-D starter kit) and loaded onto Mini-PROTEAN TGX AnyKd precast gels (Bio-Rad) and run at 200 V for 35 minutes. Gels were then rinsed in water and stained with G-250 coomassie blue stain (Bio-Rad) for 1 hr to visualise proteins. Original (unedited) gel images can be found in Fig. S2.

The first dimension, isoelectric focusing (IEF) electrophoresis, was performed using 18 cm, pH 3–10 non-linear IPG strips (Bio-Rad, Hercules, CA, USA). Each sample was diluted in rehydration buffer, which contained 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.5% ampholytes at pH 3–10 and Bromophenol blue to a final volume of 340 mL containing 250 μg protein. The IPG strip was rehydrated for 14 h at room temperature. Isoelectric focusing with Protean IEF Cell (Bio-Rad, Hercules, CA, USA) was performed at 20 °C using the following protocol: 1000 V for 2 h, 4000 V for 1 h, 8000 V for 1.5 h, and keep 8000 V for 9 h. After IEF, the IPG strip was equilibrated in two equilibration solutions for 15 min each with gentle shaking. The first equilibration solution contained 6 M urea, 2% SDS, 20% glycerol, 0.05 M Tris-HCl (pH 8.8), and 2% DTT. In the second equilibration solution, DTT was replaced by 2.5% iodoacetamide. For the second dimension, SDS-PAGE, the IPG strip was transferred to a homogeneous polyacrylamide gel (12%, 200 × 230 × 1.0 mm) and the electrophoresis was performed using a gel running system (Bio-Rad, Hercules, CA, USA) at a current of 40 mA per gel for 4 h (see Figure S2).