( les) and were vortexed between cycles ...)Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen Flavobacterium psychrophilum, Using a 2-D Electrophoretic Approach

les), and were vortexed between cycles for 12 min. Endonuclease was added to a concentration of 150 U/ml and samples were incubated at room temperature for 30 min to digest DNA. Samples were then centrifuged (12,000 x g) and supernatant containing hydrophilic proteins was retained (E1). To separate hydrophobic proteins, reagent 2 (8 M urea, 4% CHAPS, 40 mM Tris, and 0.2% Bio-Lyte 3/10 carrier ampholytes) containing 2 mM tributylphosphine (TBP) was added to the pellet. The sample was vortexed for 2 min and centrifuged as above. The supernatant, containing hydrophobic proteins (corresponding primarily to loosely bound membrane proteins), was retained (E2). Reagent 3 (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 40 mM Tris and 0.2% Bio-Lyte 3/10 carrier ampholytes) with 2 mM TBP was then added to the pellet and the process repeated to obtain highly hydrophobic proteins generally associated with tightly bound membrane proteins (E3). Samples were either subjected to isoelectric focusing immediately following extraction or stored at -80C until needed.

Biotinylation of Cell Surface Components
In order to detect cell surface membrane proteins and allow comparison to sequentially extracted proteins, viable bacteria were biotinylated prior to protein extraction. Bacteria were harvested in log-phase growth at a concentration of approximately 5.0 x 10⁷ viable bacteria/ml (0.4 absorbance) and washed twice in ice-cold PBS (pH 8.0) taking care not to disrupt or lyse cells. The bacteria were resuspended to an absorbance of 0.2 at 525 nm and 1.7 mg of sulfo-NHS-LC-biotin was added per ml of bacteria. This mixture was allowed to rock for 30 min at room temperature. The bacteria were then wa
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