Estimation of the dissociation of the light and heavy chain of the Ala221Val FV variant. The recombinant FV variants (0.5 nM) were activated with thrombin (0.5 U/mL) for 10 minutes and then left on 37°C. Subsamples were then withdrawn at different time points and immunoprecipitated with a biotinylated monoclonal antibody against the light chain (HV-1). For precipitation streptavidin-coated magnetic beads were used. FVa was eluted by boiling and analyzed on 10% SDS-PAGE under reducing conditions. The Western blots were detected with a monoclonal antibody against the heavy chain (AHV 5146). Vectastain Elite ABC kit was used to develop the Western blots. HC denotes the heavy chain of FVa, while Ab_HC and Ab_LC denote the heavy and light chain of the HV-1 antibody.