The samples were dissolved in sample buffer (70 mM LDS (lithium dodecylsulfate), 10 mM dithiothreitol, 10% (v:v) glycerol, 0.05 M Tris pH6.8) and incubated at 70°C for 10 min. The samples were loaded onto a 4–12% Bis-Tris gel PAGE gel or a 4–20% Tris PAGE gel along with a protein ladder, PageRuler Plus (Bio-Rad, Hercules, CA). Electrophoresis was carried out for 30-45min at 200 V and 400 mA. The proteins in the gels were stained using a Coomassie Brilliant Blue-based protein stain for 15 min. The gels were destained in ultra high quality (UHQ) water for 30 min, twice.
Gels were electroblotted to polyvinyldifluoride membranes using an iBlot apparatus. Membranes were blocked in TTN buffer and incubated with an RF pool diluted 1:500 in TTN for 1 h. After 3 washes in TTN, membranes were incubated with AP-conjugated GaHIgM, GaHIgA, or GaHIgG diluted 1:2000 in TTN and incubated 1 h. After further 3 washes, bound antibodies were visualized using 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue-tetrazolium (BCIP/NBT) substrate tablets.
Gels were electroblotted to polyvinyldifluoride membranes using an iBlot apparatus. Membranes were blocked in TTN buffer and incubated with an RF pool diluted 1:500 in TTN for 1 h. After 3 washes in TTN, membranes were incubated with AP-conjugated GaHIgM, GaHIgA, or GaHIgG diluted 1:2000 in TTN and incubated 1 h. After further 3 washes, bound antibodies were visualized using 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue-tetrazolium (BCIP/NBT) substrate tablets.