Lactobacillus cultures were grown overnight at 30uC, harvested and resuspended to an OD 600 of 1.0 in TGE buffer [25 mM Tris-HCl pH 8, 50 mM glucose, 10 mM EDTA pH 8]. For cell wall removal, aliquots of 1 ml were treated with 250 KU/ml of lysozyme (Lyz; Novagen, Gibbstown, NJ) for 45 min at 37uC. Protoplasts and cell wall fractions were separated by centrifugation and the supernatants containing the cell walls collected. Protoplasts were washed, resuspended in TED buffer [25 mM Tris-HCl pH 8, 1 mM EDTA, 1 mM DTT] and disrupted with a FrenchH press. Supernatants were ultracentrifugated for 1 h to separate membranes from cytosol fractions. Three (3) mg of each fraction was analyzed on 15% denaturing polyacrylamide gels, electrotransferred to PVDF filters and used for immunoblot analysis with mAb LA2.2 as described above.