Ap conjugated goat anti human igg antibody

SDS-PAGE and Western blot analyses were performed on purified (protein A) rCMG2-Fc variants. Protein was denatured and reduced by treating samples at 95°C for 5 min with 5% (v/v) of 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). For nonreducing SDS-PAGE, samples were denatured by heat treatment at 95°C for 5 min. Samples were loaded to precast 4–20% SDS-Tris HCl polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA), running at 200 V for 35 min. For SDS-PAGE, the gel was washed three times with water and stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad Laboratories, Hercules, CA). For Western blot analysis, samples were transferred to a nitrocellulose membrane by electrophoretic transfer using the iBlot Gel Transfer Device (ThermoFisher, Waltham, MA). For Western blot detecting the CMG2 domain, the membrane was probed with a goat anti-CMG2 polyclonal antibody (ThermoFisher, Waltham, MA) at a concentration of 0.3 μg/ml, followed by incubation of a polyclonal AP-conjugated rabbit anti-goat IgG antibody (Sigma-Aldrich, St. Louis, MO) at 1:10,000 dilution. For Western blot detecting the Fc domain, the membrane was incubated with a polyclonal AP-conjugated goat anti-human IgG antibody (Southern Biotech, Birmingham, AL) at 1:3,000 dilution. The blots were developed using SIGMAFAST BCIP/NBT (Sigma-Aldrich, St. Louis, MO) according to the product instruction.