Coomassie brilliant blue r 250 solution

Culture media from recruitment experiments was collected from top and bottom compartments, separately. Samples were centrifuged (1200 rpm, 10 min, 4°C), and proteins in supernatants were precipitated with acetone (1∶6, vol:vol) at −20°C, before centrifuging (14000 rpm, 5 min, 4°C) and discarding supernatants. Acetone remnants were evaporated at rt, protein pellets dissolved in 30 µl ultrapure water under agitation and protein content was quantified using Dc Protein Assay (Bio-Rad). Samples were prepared by mixing 2.25 µg of protein per sample with loading buffer (SDS 0.1%, 0.04% sucrose in Tris buffer 0.25 M, pH 6.8) and resolved in zymography gels: stacking gels-5% polyacrylamide (Bio-Rad); resolving gels-10% polyacrylamide containing 0.1% gelatin (Sigma-Aldrich), both with 0,1% SDS. Gels were incubated twice in 2% TritonX-100 (Sigma-Aldrich) for 15 min, rinsed with water and incubated in MMP substrate buffer (CaCl₂ 10 mM in Tris buffer 50 mM, pH7.5) for 16–18 h (37°C, under agitation). After, gels were rinsed with water, incubated with Coomassie Brilliant Blue R-250 solution (Sigma) in 50% methanol and 10% acetic acid (Merck, Algés, Portugal) and washed with water until clear proteolytic activity bands were seen against the Coomassie-stained, gelatin blue background.

Total protein was extracted from washed platelets and subjected to immunoprecipitation. The immunoprecipitates were eluted with SDS sample buffer, resolved on an SDS-PAGE gel, and stained with Coomassie Brilliant Blue R-250 solution (Sigma, St. Louis, MO, USA). In addition to the TLR4 band, the protein bands common to the anti-TLR4 antibody IP sample but not present in the mouse IgG IP control sample were excised from the gel. The gel pieces were then washed, reduced, alkylated, and digested with trypsin. The tryptic peptides were then analyzed by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to an Agilent 1100 HPLC (Agilent Technologies, Inc., San Jose, CA, USA). The MS/MS spectra were searched using Sequest through the Bioworks Browser version 3.3.1 (Thermo Scientific, San Jose, CA, USA) against the NCBI nonredundant protein database.

For the analysis of MMP activity, cells were treated either with vehicle or β-NEP for 12 h. Then, protein samples were prepared [51 (link)] and loaded without heating on a 10% SDS-polyacrylamide gel including 2 mg/mL gelatin from porcine skin (Sigma-Aldrich, St. Louis, MO, USA). After electrophoresis, the gels were washed in 2.5% Triton X-100 for 30 min at room temperature to allow the proteins to renature and then incubated at 37 °C overnight in the substrate buffer (50 mmol/L Tris-HCl, 200 mmol/L NaCl, 10 mmol/L CaCl₂, 1 μmol/L ZnCl₂). Proteins were stained with Coomassie Brilliant Blue R-250 solution (Sigma-Aldrich) to reveal zones of lysis, and band intensity was calculated using ImageJ software.