Colloidal coomassie brilliant blue

Bacterial cell-associated flagellin was isolated as described previously.⁸⁵ Briefly, a single bacterial colony of each group was inoculated in TB medium and cultured overnight at 37 °C at 150 rpm. After dilution to OD₆₀₀ = 0.01, 0.1% l-arabinose was added for induction of the protein, and culturing was continued for indicated time points. Flagella were sheared off by forcing the sample 15 times through a syringe with a needle of 0.51 mm diameter (BD Microlance). One ml of samples was collected, centrifuged at 17,000 rpm and the supernatant was mixed with cold trichloroacetic acid (v:v = 3:1, Sigma). Samples were incubated at −20 °C for 2 h, followed by centrifugation at 17,000 rpm for 40 min at 4 °C. The cell pellet was collected for SDS-PAGE analysis (4% stacking gel, 12% running gel) and the gel was stained by colloidal Coomassie brilliant blue (Sigma). Uncropped gels are exemplarily shown in the Supplementary Information file.

Ghost pellets were subjected to a series of freezing/thawing cycles to remove remaining haemoglobin and resuspended in 300 μL of 2-DE sample buffer (7 M Urea, 2M Thiourea, 4% CHAPS, 50 mM DTT, 1% ampholytes), loaded onto 13 cm pI 4–7 IPG strips by passive rehydration and focused at a current limit of 50 mA/IPG strip using a fast voltage gradient (8000 V max, 24,000 Vh) at 15°C. The second dimension was carried out on pre-cast 4–12% Bis-Tris Midi Protein Gels using a NuPAGE Novex system (Life Technologies) at 75 V constant voltage and 10°C. Analytical 2-D gels were transferred to PVDF membranes using an iBlot Dry Blotting System (Life Technologies) and imaged using BAS-IP SR 2040 E phosphor storage plates (GE Healthcare) for 48 hr. High-resolution imaging and accurate quantitation of ³²P-labeled protein spots was achieved using a Typhoon FLA 7000 laser-scanning detection system (GE Healthcare). Preparative 2-DE gels were stained using Colloidal Coomassie Brilliant Blue (Sigma) and spots matching ³²P-labeled protein spots manually excised and subjected to LC-MS/MS analysis.

For each cultivation conditions of the BH1.1 cultures, the cells were pelleted at 2520 × g, 8°C for 30 min. Proteins were extracted as described (Starke et al., 2017). Briefly, cells were suspended in SDS buffer (0.1 M Tris/HCl, pH 6.8, 1.25% w/v SDS, 20 mM dithiothreitol) and disrupted using the FastPrep instrument (MP Biomedicals, Santa Ana, CA, USA) and three cycles of freeze and thaw (freeze in liquid nitrogen, thaw in 40°C water bath). Samples were centrifuged (7800 g, 10 min, 4°C), and the supernatant was mixed with an equal volume of phenol solution (10 g ml⁻¹) and incubated at room temperature for 60 min. The proteins in the phenol phase were precipitated using 100 mM ammonium acetate in methanol. Dried pellets were resuspended in 20 μl of SDS sample buffer, heated at 90°C for 4 min and separated by 1D‐SDS polyacrylamide gel electrophoresis. Proteins were stained with colloidal Coomassie brilliant blue (Merck). After the gel electrophoresis run, each sample lane was cut, separated in tubes, destained, reduced and alkylated. Finally, the protein lysate was proteolytic cleaved using trypsin (Promega). Extracted peptide lysates were desalted using SOLAμ SPE‐plates (Thermo Fisher). Peptides were dissolved in 0.1% (v/v) formic acid and injected to a liquid chromatography tandem mass spectrometer (nanoLC‐MS/MS).