3 3 5 5 tetramethylbenzidine tmb ready to use solution

For electrophoretic analysis and Western blotting, venom pools from the species C. panamensis, C. granosus, C. bicolor, T. pachyurus, T. cerroazul, T. asthenes and T. festae were included. For electrophoresis analysis, a SDS-PAGE analysis, under reducing conditions, with 12.5% gels were done according to the method proposed by Laemmli (Laemmli, 1970), then protein staining was made with Coomassie Brilliant Blue. For Western Blotting analysis, venom samples were first separated by SDS-PAGE (12.5% gels), and then transferred to a polyvinylidene difluoride membrane using a transfer apparatus Owl semi-dry system for 1 h at 400 mA. After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 2 h at room temperature, the membrane was washed three times with TBST and incubated first with the rabbit IgG antibodies (1:50), washed again three times with TBST, and then the second antibody was added (goat anti-rabbit IgG coupled to alkaline-phosphatase at 1:2000). Membranes were washed once more with TBST three times and developed with 3,3′,5,5′-tetramethylbenzidine (TMB) ready-to-use solution (Invitrogen Antibodies, Thermo Fisher Scientific, Asheville, NC, USA) according to the manufacturer's protocols.