Western blot analysis of factor VIIa/tissue factor/DOPC:DOPS/Ca²⁺ enzyme complex activation of plasma factor IX and factor IXR94S using a factor IXa heavy chain antibody. Factor IXpl (A) or IXR94S (B) was activated by factor VIIa/tissue factor/DOPC:DOPS/Ca²⁺ enzyme complex in TBS at 37°C. The reaction component concentrations were: 0.5 μmol/L factor IXpl or IXR94S, 2 nmol/L factor VIIa, 4 nmol/L tissue factor, 1 mmol/L DOPC:DOPS, and 5 mmol/L Ca²⁺. The activation products were sampled at the indicated time intervals, resolved on reducing 5% to 15% SDS-PAGE, and transferred to PVDF membrane. The membrane was blotted with the C10D monoclonal antibody, which recognizes the heavy chain (HC) of factor IXa, and developed using chemiluminescence. The positions of intact factor IX and IXaHC are indicated. The positions of the molecular weight markers are indicated at left.