His-tagged Protein Immunoprecipitation

Each strain harboring the His-tagged constructs (Table 3) was grown in static overnight biofilm cultures as described above. For plasmid maintenance, ampicillin was added. Culture supernatants were subsequently collected, filtered sterilized, and TCA precipitated. The total protein pellets were resuspended in 1 ml NP-40 with protease inhibitor cocktail (Roche Diagnostics GmbH). Samples were precleared using protein A/G plus agarose beads (Pierce), followed by immunoprecipitation with a mouse anti-His affinity resin (GenScript) or a negative-control mouse IgG antibody (Santa Cruz). Samples were incubated overnight at 4°C with rotation. On the following day, protein A/G plus agarose beads were added to the negative-control IgG samples, and the mixture was incubated for 1 h at 4°C with rotation. Afterwards, the beads or resin samples were pelleted, washed six times, and boiled in acidified Laemmli lysis buffer as previously described for adherence proteins (91 (link)). After boiling, the samples were neutralized with basic Laemmli lysis buffer. Samples were run on a 4 to 20% SDS-PAGE gel (Bio-Rad), and Western blot analysis was performed as previously described (30 (link)) using a primary anti-His antibody (Qiagen) and a secondary Alexa Fluor 700 goat anti-mouse immunoglobulin antibody. The Western blots were scanned using an Odyssey infrared detection system (Li-Cor).