Interaction of recombinant FVIII domain polypeptides with FIX. To characterize recombinant FVIII domain polypeptides, a pull-down assay was performed with FIX. GST-tagged polypeptides were purified and incubated with either recombinant FIX (A) or standard human plasma (B). The interacting proteins were precipitated by incubation with a 50% slurry of glutathione-agarose beads. The proteins bound to the GST-FVIII complexes were eluted and analyzed by western blotting with anti-GST (upper box) or anti-FIX (lower box) antibodies. Specific lanes: GST pull-down with GST-FVIII-A1 (lane 1), GST-FVIII-A2 (lane 2), GST-FVIII-A3 (lane 3), and GST-FVIII-C (lane 4).