Dot blot analysis of exposed (external) and protected (internal) regions of LtxA in solution and in DMPC membranes.

(A) Dot blot analysis of 1) trypsin-exposed LtxA, 2) digested domains of LtxA-liposomes, and 3) undigested domains of LtxA-liposomes. (1) Full-length, digested LtxA was used as a control (Dot #1). Antibodies 28 and 83 were able to detect their epitopes in full-length LtxA; however, the epitope for antibody 107 was digested in the typsin treatment and therefore, undetectable. (2) LtxA was incubated with DMPC liposomes for 30 mins and then digested with trypsin. The peptides resulting from this digestion, derived from domains of LtxA residing outside of the liposome, were separated and spotted on the nitrocellulose (Dot #2). None of the antibodies detected their epitopes, indicating that the epitopes for mAbs 28 and 83 reside inside the membrane. The epitope of mAb 107 is not detected, indicating either it is digested (thus, residing outside of the liposome) or is protected inside the liposome. (3) Finally, the liposomes, containing the domains of LtxA residing inside the liposomes were spotted on the nitrocellulose (Dot #3). Antibodies 28 and 83 detected their epitopes, confirming that these two epitopes are located in a domain of LtxA that is located inside the liposome. The epitope to mAb 107 was not detected, indicating that this epitope is not located within the membrane, and must therefore be located outside of the membrane. (B) Quantification of dot blot intensities, determined using ImageJ. Each bar represents detection of LtxA by one of three monoclonal antibodies, mAb 28 (black), mAb 83 (white), mAb 107 (hashed). Each intensity is normalized by the intensity of the digested LtxA in solution dot detected by the respective antibodies.