Hrp conjugated anti goat immunoglobulins

5 μg/ml of recombinant proteins or gelatin were immobilized in PBS on MaxiSorp 96-well microtiter plates (Nunc) over night at 4°C. After washing with PBS-T, wells were blocked with 0.1% (w/v) gelatin in PBS for 2 h at RT. Wells were washed with PBS-T and incubated with 10 μg/ml plasminogen at RT for 1 h. Following three wash steps with PBS-T, 93.5 μl of a reaction mixture was added, containing 50 mM Tris/HCl, pH 7.5 and 20 μg/ml fibrinogen. To activate bound plasminogen to plasmin, 6.5 μl uPA (2.5 μg/ml) was added. Microtiter plates were incubated at 37°C and aliquots were taken at different time intervals. Reactions were stopped by addition of SDS-PAGE sample buffer and separated by 10% Tris/Tricine SDS-PAGE. Following transfer to nitrocellulose membranes, fibrinogen and its degradation products were visualized using a polyclonal goat antiserum (1:1,000) raised against fibrinogen (Acris) and HRP-conjugated anti-goat immunoglobulins (Dako) (1:1,000).

Degradation of C3b by Tuf-bound plasminogen was assayed in a fashion similar to the fibrinogen degradation assay described above. Briefly, immobilized Tuf proteins or gelatin (10 μg/ml) were incubated with plasminogen (20 μg/ml) and after several wash steps, 93.5 μl of a reaction mixture consisting of 50 mM Tris/HCL, pH 7.5 and 20 μg/ml C3b was added to the wells. Plasminogen was activated to plasmin by addition of 6.5 μl uPA (2.5 μg/ml). Microtiter plates were incubated at 37°C and aliquots were taken at the indicated time intervals. Samples were separated by 10% Tris/Tricine SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then probed with a polyclonal goat antiserum raised against human C3 (Acris) (diluted 1:1,000), followed by HRP-conjugated anti-goat Immunoglobulins (Dako) (diluted 1:1,000). Antigen-antibody complexes were visualized with TMB.