*4.6. Electrophoresis and Western Blotting*

Venom samples of 5 μg were prepared by adding non-reducing sample buffer and were separated by electrophoresis on 12% SDS-PAGE gel. Amersham ECL High-Range Rainbow Molecular Weight Markers (Cytiva, Marlborough, MA, USA) was also included. The gel was stained with Coomassie Blue or transferred to Amersham Hybond-P PVDF Membrane (Cytiva) for 1 h at 10 V using a BioRad Trans-blot SD semi-dry transfer cell. The membranes were stained with 0.5% Ponceau S, washed with ultrapure water and then blocked with PBS/5% BSA at 37 ◦C for 2 h. Next, the membranes were washed twice with PBS/0.1% Tween 20 for 20 s and were incubated overnight with an appropriate dilution of pooled antisera (Groups I, II, III and IV corresponding to 45 days after immunization) at 4 ◦C. The membranes were washed 4 times for 5 min and incubated with anti-mouse IgG (whole molecule)-HRP (Sigma) at 1/30,000 dilution in PBS/1% BSA for 1 h at room temperature. After washing, the blots were developed using Amersham ECL Prime Western Blotting Detection Reagent (Cytiva).
