Western blot analysis of human IgE was performed by loading 0.5ug of purified human IgE, IgM, IgA and IgG per well and 10ul of PageRuler Prestained Protein Ladder per well onto a 20% Tris-glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter, and blots were blocked with 5% milk in TBST for at least 1 hour at room temperature. Human IgE was detected at ~75kD using a human IgE monoclonal antibody at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse light chain secondary antibody at a dilution of 1:40,000 for at least 30 minutes at room temperature (left blot). To control for protein loading, a duplicate blot was probed with a mixture of goat anti-human IgG, IgM, and IgA and goat anti-human IgE antibodies at concentrations of 1 µg/mL, followed by a mouse anti-goat IgG-HRP secondary antibody at a dilution of 1:8000 (right blot).