Protein concentrations of the supernatant (cytosolic fraction) were measured using the colorimetric assay RC DC Protein Assay (Bio-Rad), using bovine serum albumin (BSA) as standard
protein, according to the manufacturer’s instructions. The supernatants were stored in aliquots at -80°C. Two-dimensional gel electrophoresis conditions Aliquots of the L. sakei cytosolic fraction corresponding to 50 μg (analytical gel) or 200 μg (preparative gel) of protein were diluted by adding a rehydration buffer (6 M urea (Merck), 2 M thiourea (Merck), 4% 3- [(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich), Selleck 4EGI-1 0.5% immobilized pH gradient (IPG) buffer pH 4-7 (GE Healthcare Bio-Sciences), and 2.5% dithiothreitol (DTT; Bio-Rad)) to a final volume of 380 μl. This SRT2104 solution was
used to rehydrate 18-cm pH 4-7 linear IPG strips (GE Healthcare BioSciences). Strips were passively rehydrated at room temperature for 12-16 h under mineral oil, before isoelectric focusing (IEF) was performed in an Ettan IPGphor II unit (GE Healthcare Bio-Sciences, Uppsala, Sweeden) as follows: 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, from 1000 to 8000 V in 30 min, and finally 8000 V for 6 h. AZD8931 price The strips were incubated at room temperature for 15 min in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/l) glycerol (Merck) and 2% (w/v) sodium dodecyl sulfate (SDS; Shelton Scientific)) supplemented with 1% (w/v) DTT, followed by 15 min in equilibration buffer containing 2.5% (w/v) iodoacetamide (Merck). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide gels was carried out with an Ettan DALT II system (GE Healthcare Bio-Sciences, Uppsala, Sweeden). Proteins were resolved at 20°C at a current of 2.5 mA/gel for 45 min and then at 25 mA/gel until the tracking dye had migrated to the bottom of the gel. Analytical gels were silver stained as described by Blum et al. [37] and preparative gels according to Shevchenko et al. [38]. For the final analysis, three 2-DE PI-1840 gels were
run from each strain from each of the two independent bacterial cultures. Image and statistical analysis Digitized 2-DE images (16-bit greyscale, 300 dpi) of the stained gels were acquired with an office scanner (Epson Perfection 4990 Photo, Epson) and imported into Progenesis SameSpots software v.3.1 (Nonlinear Dynamics). For each strain, five glucose images and five ribose images were aligned using one selected glucose image as a reference [39]. Spots were detected simultaneously across the images leading to one spot map, an approach which addresses the problems of missing values and reduces variance in spot volume across biological or technical replicates by applying the same spot outline across the image series [39, 40]. The spot pattern was manually edited, gel artefacts were removed, and images were grouped glucose vs. ribose.
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