( tify and further characteriz...)Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen Flavobacterium psychrophilum, Using a 2-D Electrophoretic Approach

tify and further characterize antigens of interest. The role these play in pathogen virulence and immunoprotection is currently being evaluated using the PROTEAN IEF cell and ReadyStripTM IPG strips to isolate and excise specific bacterial proteins. Sequential extraction procedures are being utilized along with biotin labeling to distinguish between cytoplasmic, membrane-associated, and cell surface proteins. Since fish are capable of mounting a protective immune response against F. psychrophilum, it is speculated that a 2-D approach will be valuable in allowing identification and characterization of immunodominant and virulence-associated proteins in relation to specific bacterial strains.

Methods
Bacterial Culture
Virulent (CSF-259-93) and nonvirulent (ATCC 49418) isolates of F. psychrophilum were cultured in tryptone yeast extract salts broth at 15C. Bacteria used for 2-D PAGE were harvested from log-phase cultures and adjusted to an absorbance of 0.4 at 525 nm. This corresponds to an approximate bacterial concentration of 5 x 10⁷ cfu/ml.

Sequential Extraction of Bacterial Proteins
To identify hydrophilic (primarily cytoplasmic nonmembrane proteins) and hydrophobic proteins associated with cell or organelle membranes, a method for sequential extraction was developed using the ReadyPrepTM sequential extraction kit. Briefly, proteins were extracted from F. psychrophilum (strain CS-259-93) following harvest from log phase cultures. Samples were washed twice in PBS and resuspended to an approximate protein concentration of 12 mg/ml in reagent 1 (40 mM Tris, pH 8.0). Bacteria were lysed by rapid freezethawing (10 cyc
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