Figure 3.

Representative experiment showing patient autoantibody binding to Gla‐domain free PS. SDS‐PAGE was performed using 10% polyacrylamide gel. Thrombin‐treated PS or PS under reducing conditions (5 μL of 10 μg mL⁻¹ solution) was applied to each lane. Transfer to PVDF membrane was done for 20 min at 0.1 amps. Membranes were blocked for 2 h with 1% BSA in TBS at pH 7.3. Incubation with mouse polyclonal antibodies (PoAb) to PS, normal plasma (control), or anti‐PS‐positive patient plasmas (1/100) was done for 1.5 hour followed by three washes with 0.05% Tween 20/TBS. The membrane was exposed to horseradish peroxidase‐conjugated anti‐mouse immunoglobulins or polyclonal antibodies to human IgG or IgM for 1 hour. Gla, γ‐carboxyglutamic acid; PS, protein S; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin; TBS, Tris‐buffered saline