Immunoblot detection of proteins released in free-soluble form by biofilm and planktonic A. actinomycetemcomitans strains, using polyclonal antisera raised against LtxA, PAL, GroEL, and CRP (lysis marker), respectively. Proteins released from strain D7S to PBS through the 20 nm filters of the inserts were collected after 6 h of incubation and a concentrated sample (denoted 1) equal to 0.75 ml filtrate was applied on the gels where indicated. The following whole cell preparation samples (10 μg protein each) were loaded as controls where indicated: 2. D7S, 3. D7S ΔcdtABC/ltxA, and 4. JP2 (high producer of LtxA ). The sizes of the proteins (kDa) in the prestained molecular weight marker (M) are indicated. The reactive band corresponding to each protein is indicated with an arrow.