In the case of the mutants d8-60a, d8-60b, d8-60c, all three generated identical length PCR products by this method indicating identical deletion
end points. Membrane protein analysis The outer membrane proteins (OMPs) were extracted as previously described  using equal number of cells selleck kinase inhibitor (equivalent to 5 ml of cells diluted to an OD600 of 5.0). The membrane pellet was resuspended in 200 μl of SDS sample buffer containing 5 mM tributylphosphine and 20 mM acrylamide for reduction and alkylation of proteins . The solubilized proteins were diluted 1:5 in SDS sample buffer and 5 μl subject to polyacrylamide gel electrophoresis using a Criterion XT precast gel (4-12% Bis-Tris; Bio-Rad). Protein gels were stained with Flamingo protein stain (Bio-Rad) and imaged using a Pharos FX Plus Molecular Imager (Bio-Rad).
Flamingo stained protein gels were post-stained with colloidal Coomassie G-250 stain and proteins of interest excised for identification by www.selleckchem.com/products/wzb117.html LC-MS/MS as previously described . PEAKS software (Bioinformatics Solutions Inc.) was used to directly search peptides against a protein sequence FASTA output derived from the V. rotiferianus DAT722 genome . The highest PEAKS score (percentage based on a p-value < 0.05) was taken as the closest peptide match. The full sequence of identified proteins is given in the additional file 1. Acknowledgements This work was supported by a grant from the National Health and Medical Research Council of Australia. ML is supported by an ithree Institute Postdoctoral Fellowship. Electronic supplementary material Additional file 1: lists the full sequence of outermembrane proteins that showed changes in concentration between wild type DAT722 and the mutant d8-60a under particular growth conditions. Proteins were identified
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