Detecting GPIHBP1 autoantibodies with western blots

The medium from Drosophila S2 cells expressing rhGPIHBP1 was size-fractioned on 12% Bis-Tris SDS-PAGE gels, then transferred to a nitrocellulose membrane. The membrane was blocked in Odyssey blocking buffer (Li-Cor). Membranes were then incubated with the plasma from patient A1 (20 AU of GPIHBP1 autoantibody/ml). The binding of autoantibodies to GPIHBP1 was detected with an IRDye680–donkey anti-human IgG (1:200). rhGPIHBP1 was also detected with an IRDye800-conjugated monoclonal antibody against human GPIHBP1 (RF4)¹⁷ (1:500).