Nupage 3 8 tris acetate

Example 5

Plasmin Digestion of α-ext rhFib and Plasma Derived Fibrinogen

Fibrinogenolysis of purified recombinant human α-ext rhFib was tested by incubation with plasmin. Briefly, fibrinogen was diluted in TBST (50 mM Tris-HCl, pH7.4, 100 mM NaCl, 0.01% Tween-20), CaCl2 or EDTA was added (5 mM final concentration) and plasmin was added (10 nM final concentration), followed by an incubation at 37° C. At several points in time samples were taken and mixed immediately with SDS-PAGE sample buffer (NuPAGE LDS sample buffer, Invitrogen, cat# NP0007). Samples were then subjected to size separation on a non-reduced SDS-PAGE gel (NuPAGE 3-8% Tris-Acetate, Invitrogen, cat# WG1602). Protein was visualized by Coomassie staining (SimplyBlue SafeStain, Invitrogen, cat# LC6060).

The results, as shown in FIG. 3, indicate that the plasmin mediated digestion of α-ext rhFib is significantly slower than for plasma derived fibrinogen. For example, in the presence of Ca2+, after 60 minutes all of the plasma derived fibrinogen is degraded down to fragment D and E species. For α-ext rhFib this takes more than 120 minutes and only the overnight incubation shows substantial amounts of fragment E generation, which are already present in the digest of the plasma derived fibrinogen after 30 minutes.