Cross-linking of factor VIIIa with EDC. Factor VIIIa (750 nmol/L) was incubated in the absence (lane 1) and presence of EDC at 75 μmol/L (lane 2), 188 μmol/L (lane 3), 375 μmol/L (lane 4), 750 μmol/L (lane 5), 1.5 mmol/L (lane 6), or 3 mmol/L (lane 7) for 2 hours at room temperature in a reaction (35 μL) containing 25 mmol/L Mes, pH 6, and 0.01% Tween-20. An aliquot was removed from each sample and assayed for factor VIIIa activity (C). SDS sample buffer was added to the remainder of each sample and reactions were subjected to electrophoresis on 6% to 15% gels, transferred to PVDF, and immunoblotted with the anti-A1 subunit antibody, 58.12 (A), or the anti-A2 subunit antibody, R8B12 (B).