Ettan ipgphor isoelectric focusing system

The serum samples were subjected to two-dimensional gel electrophoresis. Briefly, 100 μg of each serum sample was loaded onto an immobilized pH gradient (IPG) strip (GE Amersham, United Kingdom) with pH variation from 3 to 10. The first dimensional isoelectric focusing was implemented as follows: 30 V for 12 h, 500 V for 1 h, 1,000 V for 1 h, 8,000 V for 8 h, and 500 V for 4 h using Ettan IPGphor Isoelectric Focusing System (GE Amersham, United Kingdom). The proteins after iso-electrophoresis were separated with second dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using Hofer SE 600 (GE Amersham, United Kingdom). The second-dimension gel electrophoresis was performed on the 12.5% SDS-polyacrylamide slab gels at 15 mA for 30 min and 30 mA until the trace of bromophenol blue was 0.5 cm away from the bottom of the gel.

Two-dimensional electrophoresis (2-DE) was conducted according to Hu et al. (2014c (link)). The protein samples (500 μg) were loaded onto Immobiline DryStrips (18 cm long, pH 4–7, GE Healthcare) during the rehydration step at room temperature for 12 h. Isoelectric focusing (IEF) was performed using an Ettan IPGphor isoelectric focusing system (GE Healthcare) as follows: 300 V for 1 h, 500 V for 1 h, 1000 V for 1 h, a gradient to 8000 V for 4 h, and kept at 8000 V for a total of 80,000 volt-hours (Vh) at 20°C. After IEF, the focused strips were equilibrated in equilibration buffer as described by Hu et al. (2014a (link)). For the second dimension electrophoresis, the proteins were separated on 15% SDS polyacrylamide gels. Subsequently, the gels were stained using Coomassie Brilliant Blue (CBB) R-250. The 2-DE gel images were acquired with an image scanner (Uniscan M3600, China) and analyzed using the PDQuest software (Version 8.01, Bio-Rad, Hercules, CA, USA). Protein spots with significant (>2-fold change) and reproducible changes were selected for MS analysis.

Precast IPG strips (13 cm, pH 3–10, linear, GE Healthcare) were rehydrated with 250 µL of DeStreak rehydration solution (GE Healthcare) containing 1% IPG buffer (GE Healthcare) and 100 µg of B. lanceolatus venom for 16 h at room temperature. First dimension separation was carried out using an Ettan IPGphor Isoelectric Focusing System (GE Healthcare), following th emanufacturer’s instructions. We followed a five-phase electrophoresis program; a step of 500 V for 60 min, a gradient of 1000 V for 60 min, a gradient of 8000 V for 2 h 30 min, a step of 1000 V for 12 h (so as to run overnight), and finally a step of 8000 V for 30 min. Right at the end of the separation, the IPG strips were sequentially incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 2% SDS, 6 M urea, 30% glycerol, bromophenol blue) containing 10 mg/mL DTT for 15 min and then in equilibration buffer containing 25 mg/mL iodoacetamide for 15 min. After that, they were briefly washed in electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) and applied to 12% SDS-polyacrylamide gels (10 × 10 cm) for second dimension electrophoresis, using the Hoefer Mini VE apparatus (GE Healthcare). The gels were finally silver-stained.