Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS PAGE)

The degradation profile after fibrinolysis was analyzed using SDS-PAGE. We mixed SDS PAGE loading buffer containing BME (Biorad, Cat# 610737) with each sample in a 1:1 (v/v) ratio and incubated at 95 °C for 10 min. We loaded the samples onto the wells of 8% Tris.glycine gels. We ran electrophoresis in Tris.glycine buffer (Biorad, Cat# 1610732) for 30 min at 50 V, followed by 1 h at 120 V. We used the precision plus protein kaleidoscope pre-stained protein standards (Biorad, Cat# 1610375) as molecular weight markers. The SDS-PAGE gel was stained with Coomassie Brilliant Blue R-250 (Biorad, Cat# 161-0436) and imaged using a digital camera after destaining.

For fibrin without crosslinks, we prepared fibrin samples in 1.5 mL Eppendorf warmed to 37 °C by adding 40 µL of 25 mg/mL fibrinogen (Cayman chemical, Cat# 16088), 20 µL of 1000 U/mL thrombin (Cayman chemical, Cat# 13188), and 40 µL of 0.01 M PBS. The mixture became cloudy and visible threads formed immediately after the addition of thrombin. Samples were vortexed for 1 s and then centrifuged quickly for 3 s at 10,000 rpm to collect all the samples to the tube's bottom. We incubated samples at 37 °C without shaking for 15 min. Samples were then treated for 18 h either with 100 µL of 2.5% Triton buffer (2.5% Triton X-100 dissolved in 50 mM Tris, 100 mM NaCl, pH 9.0), 100 µL of 0.12 mg/mL active MMP1, 100 µL of 0.12 mg/mL inactive MMP1, and 100 µL of 0.14 mg/mL MMP9. The next morning, all four samples were prepared for SDS PAGE by 1:1 dilution in sample buffer with β-mercaptoethanol (Sigma-Aldrich, Cat# M3148) prepared from 2X Laemmli buffer (Biorad, Cat# 161-0737). We boiled the samples for 10 min after adding the dye and loaded 30 μL of each sample into the wells of an 8% polyacrylamide gel. The gel was run in 1X SDS running buffer at 70 V. After 30 min, we increased the voltage to 120 V, and gels were allowed to run for an additional 1 h. Gels were removed from the running tank and stained in 100 mL of stain prepared using 30% ethanol, 10% acetic acid, and 0.5% Coomassie Brilliant Blue R-250 (Biorad, Cat# 161-0436). The gel was stained for 2 h and washed twice in DI water to remove excess dye, followed by destaining in 200 mL of 30% ethanol and 10% acetic acid for 24 h. We imaged the gel using a digital camera.