Bacterial Protein Extraction and Purification

Lysis was carried out using a Q Proteome Bacterial Protein Prep kit (Qiagen srl; Milan, Italy) containing 100 mg/mL lysozyme. Benzonase 0.25 U/µL (Sigma-Aldrich srl; Milan, Italy) was then added to degrade nucleic acids. Samples were incubated at room temperature on ice, then centrifuged (14,000× g, 30 min, 4 °C); the supernatant was collected in polypropylene vials (Nalgene) and newly centrifuged (50,000× g, 30 min). Aliquots of the supernatant underwent to SpeedVac (mod. SC110-SAVANT) and were finally stored at −80 °C until protein purification. Before purification, the three samples obtained by independent extractions were pooled for planktonic and biofilm samples [15 (link)]. Samples were purified using a Ready Prep 2D Cleanup kit (Bio-Rad; Milan, Italy), and the protein precipitates were treated with 50 µL solubilization buffer for two-dimensional gel electrophoresis (2-DE) (2 M thiourea, 7 M urea, 50 mM DTT, 4% CHAPS, 0.2% Bio-Lyte 3/10 ampholyte, and 0.002% bromophenol blue). After protein resuspension, each sample was centrifuged at 12,000× g, and the clear phase was transferred to a new vial where the buffer was added to yield a final volume of 450 µL. Protein concentration was determined, according to Lowry, by RC-DC Protein Assay (Bio-Rad). Concentrations of 8 and 16 µg/µL were obtained for planktonic and biofilm samples, respectively. Samples were finally stored at −20 °C until needed.