Monoclonal antibodies against chicken tropomyosin isoforms: production, characterization, and application

Eight mouse monoclonal antibodies, CH1, CH106, CH291, CL2, CG1, CG3, CG beta 2 and CG beta 6, against chicken tropomyosin isoforms have been prepared and characterized. The antigens recognized by these isoform-specific monoclonal antibodies were identified by both solid-phase radioimmunoassay and protein immunoblotting. To some extent, most antibodies showed isoform-specific, but one (CG3) recognized all isoforms of tropomyosin from chicken materials. The effects of monoclonal antibodies on the binding of cardiac tropomyosin to F-actin were investigated. Antibodies CH1, CH106, and CH291 had the ability to interfere with the binding of tropomyosin to F-actin, whereas others appeared to have no effect. Monoclonal antibody CL2 was able to distinguish the skeletal muscle tropomyosin-enriched microfilaments from the fibroblastic tropomyosin-enriched microfilaments of differentiating muscle cells. This antibody will be most useful for studying the compartmentalization of microfilaments and microfilament-associated proteins, particularly actin and tropomyosin isoforms during muscle differentiation. Immunofluorescence microscopy with CG1 antibody which recognized CEF tropomyosin isoforms 1 and 3 revealed the continuous staining of stress fibers in some populations of CEF cells. On the other hand, both periodic fluorescent staining and continuous staining of stress fibers were observed with CG3 antibody in all CEF cells.