( shed in ice-cold PBS and resuspended to...)Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen Flavobacterium psychrophilum, Using a 2-D Electrophoretic Approach

shed in ice-cold PBS and resuspended to an approximate protein concentration of 12 mg/ml in reagent 1. Washed cells were subjected to the initial extraction step (see above) and an equal volume of reagent 3 was added to obtain total proteins for 2-D PAGE separation. Following 2-D PAGE and transfer of separated proteins to a nitrocellulose membrane, biotin-labeled proteins were detected by probing blots with ExtrAvidin-alkaline phosphatase (Sigma) and visualized colorimetrically by the addition of NBT/BCIP.

IEF and Second-Dimension Separation of Proteins
Sequentially extracted or total protein samples were applied to ReadyStrip IPG Strips pH 47 or pH 310 and passively rehydrated overnight in a humidity chamber. Once fully rehydrated, isoelectric focusing of proteins was carried out with the PROTEAN IEF cell using a preset method that allowed a minimum of 20,000 V-hr to be obtained while maintaining a constant temperature of 17C. Focused strips were stored at -80C until second-dimension PAGE could be performed.

For second-dimension separation, IPG strips were equilibrated in an equilibration buffer (6 M urea, 2% SDS, 0.375 M Tris, 4% glycerol, 5 mM TBP, 2.5% acrylamide monomer) for 20 min at room temperature. Strips were then transferred to the prep wells of Ready Gel1020% Tris-HCl precast gels and electrophoresed in the Mini-PROTEAN 3 cell for 20 min at 4 mA/gel and then 1.5 hr at 12 mA/gel. Separated proteins were silver stained or transferred to nitrocellulose for detection of biotin-labeled cell surface proteins.

Results
Sequential Extraction and Biotin Labeling of Bacterial Proteins
Using the CSF-259-93 reference strain, th
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