Coomassie brilliant blue r

The activity of proteolytic enzymes was evaluated using gelatin, following the electrophoretic separation of lysates from the BMMs culture, with or without 1 μM of 2N1HIA, in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 5 days. Briefly, cells (6 well plate) were mixed with a homogenization buffer (50 mM Tris-HCl, pH 6.8, 150 mM NaCl, and 1% Triton X-100) and the lysates were collected following centrifugation (5000× g, 10 min, 4 °C). Thirty micrograms of unheated and non-denatured protein were subjected to 0.1% gelatin (Sigma) containing SDS-polyacrylamide gel electrophoresis. Gels were washed in a renaturing buffer (2.5% Triton X-100) and incubated in a Novex zymogram developing buffer (Invitrogen, Carlsbad, CA, USA) for 16 h at 37 °C. Gels were stained with 0.2% Coomassie brilliant blue R (Sigma, St. Louis, MO, USA) for 1 h, and de-stained in 20% methanol and 10% glacial acetic acid.

Reactions were prepared in 50 mM Tris base, pH 7.5. GzmB (200 nM; Emerald BioSystems) was incubated with VTI-1002 (50 µM, generous gift from viDA Therapeutics, Inc.) for 30 min at room temperature prior to the addition of either FBN (1.0 µg; Sigma-Aldrich, St. Louis, MO, USA) or DCN (1.0 µg; R&D Systems, Minneapolis, MN, USA) substrate. Reactions were incubated overnight at 37 °C in a water bath. The next day, 6× Laemmli loading buffer was added to each sample and the samples denatured at 95 °C for 5 min in a heat block. Solutions were collected by centrifugation and the samples separated on a 10% polyacrylamide gel under denaturing conditions. Gels were washed 2 × 5 min each in distilled water and stained at room temperature for 60 min in Coomassie Brilliant Blue R (Sigma) with gentle shaking. Destaining was accomplished using a solution of 45% (v/v) methanol and 10% (v/v) glacial acetic acid. Destaining solution was replaced every 30 min until proteins were easily visible. Images were collected using the Licor Odyssey Scanner under the 700 nm channel and processed using Image Studio (Licor Biotechnology, Lincoln, NE, USA).

BSA (M_w 66 kg/mol), HPβCD (average M_w ~ 1540 g/mol), sodium phosphate, and Coomassie brilliant blue R were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bradford protein assay dye reagent and acrylamide were acquired from Bio-Rad Laboratories (Hercules, CA, USA). Glacial acetic acid and ortho-phosphoric acid were obtained from Merck KGaA (Darmstadt, Germany). PageRuler™ prestained protein ladder (10 to 180 kDa) was purchased from Thermo Scientific (Waltham, MA, USA). Methanol was obtained from Anaqua Global International (Cleveland, OH, USA). Ultrapure water used was purified by Barnstead NANOpure Diamond™ water system with a 0.2 µm filter (APS Water Services, Van Nuys, CA, USA). All solvents and reagents were of analytical grade or better unless otherwise specified.

Flagellin levels were determined as described previously^{41 (link)}. The bacterial suspension washed from the LB agar plate with PBS (10 ml, OD_600nm = 0.6) was used to prepare flagellin by vigorous vortexing and subsequent centrifugation. Flagellin in the supernatant was precipitated with trichloroacetic acid (5%, w/v), separated on 12% SDS-PAGE and stained with Coomassie Brilliant Blue R (Sigma-Aldrich B7920).

The activity of proteolytic enzymes was evaluated using gelatin or casein zymography following the electrophoretic separation of lysates from young and old human gingival tissues (n = 3 individuals per group; S1 Table). Briefly, gingival tissues (30 mg) were mixed with homogenization buffer (50 mM Tris-HCl, pH 6.8, 150 mM NaCl, and 1% Triton X-100) and homogenized with a pestle on ice, and lysates were collected by centrifugation (5,000 × g, 10 min, 4°C). Twenty micrograms of unheated and non-denatured protein were subjected to 0.1% gelatin (Sigma) or 0.1% β-casein (Sigma)-containing SDS-polyacrylamide gel electrophoresis. Gels were washed in renaturing buffer (2.5% Triton X-100) and incubated in Novex zymogram developing buffer (Invitrogen, Carlsbad, CA, USA) for 16 h at 37°C. Gels were stained with 0.2% Coomassie brilliant blue R (Sigma) for 1 h and then destained in 20% methanol and 10% glacial acetic acid. gelatinolytic and caseinolytic activities were detected as white areas against a blue background. The areas were quantified with the CS Analyzer 4.0 software (ATTO Co., Tokyo, Japan).