Imagemaster 2d platinum v 6

Cells in the exponential growth phase were harvested from BHI broth, washed in Tris-HCl 50 mM (pH 7.5) and used to generate proteins extracts as described by De Angelis et al. (2001) (link). Equivalent amounts of total protein (60 μg for analytical runs or 200 μg for preparative runs for protein identification) were used for each electrophoretic run. The 2-DE was performed essentially as described by Görg et al. (1988) (link) and Hochstrasser et al. (1988) (link) using a Pharmacia 2-D-Electro Focusing (EF) system (GE Healthcare, Milano, Italy). Gels were stained using Brilliant Blue G-Colloidal Concentrate (Sigma) or an MS-compatible silver method. The protein maps were scanned using LabScan on an ImageScanner (GE Healthcare) and were analyzed using ImageMaster 2D Platinum v.6.0 (GE Healthcare). Three gels from three independent experiments were analyzed, and the spot intensities were normalized (De Angelis et al., 2001 (link)), with the spot quantification for each gel calculated as a relative volume (% vol) that corresponded to the volume of each spot divided by the total volume over the entire image. The comparison between different conditions for the amount of the same protein was carried out as the rate of the relative volume of the same spot found in control (untreated cells) and ethanol or other antimicrobials treated cells (Siragusa et al., 2014 (link)).

The E. coli BL21(DE3) proteome was analyzed to predict membrane‐associated and periplasmic proteins. A meta‐prediction pipeline that employs different subcellular localization prediction tools in three phases of positive and negative selections was used (Hisham & Ashhab, 2018). The identified proteins were subsequently assessed for their biological function and classified (essential vs. non‐essential) according to the ‘Keio collection’ (Baba et al., 2006), and EcoCyc E. coli database (Keseler et al., 2017), respectively. Oligonucleotides for guide DNAs, donor DNAs and primers were designed using Serial Cloner and Benchlink and BLAST was used for alignments (Altschul et al., 1990). Densitometry was performed with Image Studio Lite Version 5.2 (Li‐Cor Biosciences, Lincoln, NE USA) and for 2‐DE map analysis, ImageMaster 2D Platinum v.6.0 software was used (GE Healthcare, Chicago, IL USA).

Image analysis was performed on analytical 2D gels using ImageMaster 2-D Platinum v6.0 (GE Healthcare). For each condition tested, image analysis was performed on three different spot maps from three biological replicates for a total of nine analyzed gels. An intra-class quality and experimental control was performed by comparing the gels and then a differential inter-class analysis was performed to detect any statistically significant quantitative and qualitative differences. Based on a fold change of at least ±2 in relative volume (%V) ratio, and on statistically analysis as reported in the tables, differentially expressed proteins were detected among the three conditions examined.