Acrylamide bis gel

The four OTA-mAbs generated and the rabbit hyperimmune polysera were purified by the ammonium sulfate precipitation method and protein A column affinity chromatography. Different fractions obtained during the purification of IgG were subjected to gel electrophoresis to check for purity. Using a mini-PROTEAN II Electrophoresis Cell (Bio-Rad, India), samples in lysis sample buffer (containing 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.01% bromophenol blue and 62.5 mM Tris-HCl, pH 6.8) were applied to a 12% acrylamide-bis gel (Bio-Rad, India) and proteins were separated by SDS-PAGE in Tris/Glycine/SDS running buffer, pH 8.3 [containing 25 mM Tris-base, 192 mM glycine, and 0.1% (w/v) SDS] for 45 min with a constant voltage of 200 V. Proteins were stained for 30 min with 0.1% Coomassie blue R-250 in 40% methanol (MeOH) and 10% acetic acid (HOAc); subsequently the gel was destained with several changes of 40% MeOH/10% HOAc for 1–3 h.

The four OTA-mAbs generated and the rabbit hyperimmune polysera were purified by the ammonium sulfate precipitation method and protein A column affinity chromatography. Different fractions obtained during the purification of IgG were subjected to gel electrophoresis to check for purity. Using a mini-PROTEAN II Electrophoresis Cell (Bio-Rad, India), samples in lysis sample buffer (containing 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.01% bromophenol blue and 62.5 mM Tris-HCl, pH 6.8) were applied to a 12% acrylamide-bis gel (Bio-Rad, India) and proteins were separated by SDS-PAGE in Tris/Glycine/SDS running buffer, pH 8.3 [containing 25 mM Tris-base, 192 mM glycine, and 0.1% (w/v) SDS] for 45 min with a constant voltage of 200 V. Proteins were stained for 30 min with 0.1% Coomassie blue R-250 in 40% methanol (MeOH) and 10% acetic acid (HOAc); subsequently the gel was destained with several changes of 40% MeOH/10% HOAc for 1–3 h.