FIG. 4.

Localization of CwlJ-His tag in spore extracts of various strains. The insoluble fraction of the extract from 1 mg of spores of strains PS3449 (cwlJ-His tag) (lanes 1), KB33 (cwlJ-His tag ΔywdL) (lanes 2), KB29 (ΔywdL) (lane 3), and KB34 (cwlJ-His tag ywdL16) (lane 4) was run on an SDS-12.5% polyacrylamide gel. Each lane contains protein from 1 mg (dry weight) of spores. The gel was blotted onto an Immobilon-P membrane (Millipore) that was hybridized with anti-His tag monoclonal antibody (Novagen) as outlined in Materials and Methods. An extract from spores of strain KB29 (lane 4) that lacks the CwlJ-His tag served as a negative control for the anti-His tag antibody. The arrow indicates an immunoreactive product at ∼50 kDa (with no His tag attached) that has been seen previously (5) and that we use here as a rough indication of protein levels. The CwlJ-His tag runs as a wide band of ∼18 kDa as shown previously (5). Only a portion of the Western blot is shown, and the numbers shown at the left indicate the migration positions of protein molecular mass markers in kilodaltons. The same experiment was run for the soluble fraction of the spore extracts, where CwlJ-His tag was not found (data not shown), as reported previously (5).