Detection of CPN and TAFIa in crude and purified serum and plasma fractions containing SDF-1α peptidase activity. Protein samples from the purification of SDF-1α peptidase activity from human serum or plasma as described in Tables 1 and 2 were electrophoresed, transferred to nitrocellulose, and immunoblotted in panels A and C with rabbit polyclonal antibody against the active subunits of CPN, and in panels B and D with a mouse monoclonal antibody against TAFI. (A-B) Lane 1: human serum (1.0 μg); lane 2: 40% ammonium sulphate pellet (1.0 μg); lane 3: anion exchange fraction eluted with 0.25 M NaCl (1.0 μg); lane 4: pooled lysine Sepharose fraction eluted with 150 mM NaCl and 150 mM NaCl containing 100 μM SDF-1 peptide (1.0 μg). (C-D) Lane 1: human plasma (0.25 μg); lane 2: 50% ammonium sulphate pellet (0.25 μg); lane 3: anion exchange fraction eluted with 0.25 M NaCl (0.25 μg); lane 4: pooled lysine Sepharose fraction eluted with 150 mM NaCl and 150 mM NaCl containing 100 μM SDF-1 peptide (0.25 μg). Bands were visualized using Promega stabilized alkaline phosphatase substrate following incubation with secondary antirabbit or antimouse antibodies linked to alkaline phosphatase. The locations of the active subunits of purified CPN (0.3 μg) and the active (TAFIa) (0.01 μg) and inactive subunits of TAFI (0.01 μg) are indicated.