Coomassie brilliant blue r 250 solution

Porphyromonas cell suspensions (10⁷ CFU/reaction), sBHI media controls, or Porphyromonas cell-free supernatants (240 µg of total protein) were incubated with 120 µg of human fibrinogen (Sigma-Aldrich, St. Louis, MO). Reaction mixtures were incubated at 37 °C under anaerobic conditions or in an atmosphere of 5% CO₂ for cell suspension or cell-free supernatants, respectively. Samples from each time point (cell suspensions: 0, 2, 18, 24 h; supernatants: 0, 2, 24, 48 h) were collected, mixed 1:1 with Novex^TM 2X sample buffer (Invitrogen) with dithiothreitol (DTT, Fisher Scientific), heated at 95 °C for 10 min, and separated on Novex^TM 10% Tris-Glycine polyacrylamide pre-cast gels (Invitrogen) at a constant voltage of 180 V. Gels were stained in 0.25% Coomassie brilliant blue R-250 solution (Fisher Scientific) and de-stained in 5% methanol/7.5% acetic acid in distilled water. Fibrinogen degradation was evaluated qualitatively by visualization of fibrinogen α chain (63.5 kDa), β chain (56 kDa), and γ chain (47 kDa) between experimental and control samples over the time-course. Gels are derived from the same experiments and were processed in parallel. Unprocessed scans of fibrinogen degradation protein gels are available in the Supplementary Information (Supplementary Figs. 9, 10).

The total protein content of Porphyromonas cell-free supernatants was determined using a bicinchoninic acid microplate assay (BCA; Pierce, Rockford, IL). Cell-free supernatants were diluted to 8 mg/mL and 5 µL of the sample was combined with 5 µL of Novex^TM Tris-Glycine SDS Sample Buffer (Invitrogen) to load 40 µg per well. Samples were separated on Novex^TM 10% Zymogram Plus (Gelatin; Invitrogen) protein gels at a constant voltage of 125 V in Novex^TM 1X Tris-Glycine Running Buffer (Invitrogen). After separation, gels were incubated in Novex^TM 1X Renaturing Buffer for 30 min at room temperature with gentle agitation, followed by two consecutive incubations in Novex^TM 1X Developing Buffer: room temperature for 30 min and 37 °C for 16 h. Gels were then stained in Coomassie brilliant blue R-250 solution (Fisher Scientific) and de-stained in 5% methanol/7.5% acetic acid in distilled water. The unprocessed scan of the zymogram gel is available in the Supplementary Information (Supplementary Fig. 8).

Rat livers were homogenized in a radio-immuno-precipitation assay (RIPA) buffer (20Mm Tris-HCl, 150mM NaCl, 1% NP-40, 0.25% Na-deoxycholate), that was supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland) in a 1:12 ratio. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, IL, USA) according to the manufacturer's recommendations. 100μg proteins were analyzed per each sample by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by fixation (methanol: acetic acid: H₂0 in a ratio of 50:10:40) and staining with Coomassie Brilliant Blue R-250 solution (Fisher BP101-25).