Silver staining was performed to establish equivalent loading of purified proteins using the Pierce™ Silver Stain Kit ( Product # 24612) (Fig b). The blot was probed with Goat anti-Mouse IgM (Heavy chain) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 ( Product # A-21044, 1:10000 dilution) and detected using theiBrightFL1500 ( Product # A44115). Resolved proteins were then transferred onto a nitrocellulose membrane( Product # IB23001) byiBlot® 2 Dry Blotting System ( Product # IB21001). Purified protein (100 ng) of Mouse IgG (Lane 1), Mouse IgM (Lane 2),Rabbit IgG(Lane 3), Rat IgG (Lane 4), Goat IgG (Lane 5) Human IgG (Lane 6), Human IgM (Lane 7) and Chicken IgY (Lane 8) (Fig a) wereelectrophoresed usingNuPAGE™ 4-12% Bis-Tris Protein Gel ( Product # NP0322BOX). Western blot was performed using Goat anti-Mouse IgM (Heavy chain) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 ( Product # A-21044) and ~70 kDa bandcorresponding to Mouse IgM Heavy Chain was observed in Mouse IgM and Human IgM, but not in Mouse IgG, Rabbit IgG, Rat IgG, Goat IgG, Human IgG and Chicken IgY.
