Fig. 4. Characterization of the conformation-sensitive anti-myosin mAb F59. (A) Stained 6% SDS-PAGE pattern of purified chicken pectoralis myosin (1), myosin from infected (2), and uninfected (3) C2C12 cells. The expressed GFP-myosin and the endogenous C2C12 myosin are resolved and migrate at 250 kDa and 220 kDa respectively. (B) Western blot of the same samples probed with anti-S1 mAb F59. F59 reacts with all three striated muscle myosin types. (C) Native myosin binding assay with anti-myosin mAbs F59 and F18. Synthetic myosin filaments (circles) or a rigor complex of acto-S1 (triangles) were incubated with Fab fragments of F59 (open symbols) and F18 (filled symbols). The free and bound Fab fragments were separated by sedimentation of the myosin-containing filaments and quantitative SDS-PAGE and densitometry. The smooth curve is the fit to a standard binding isotherm (Winkelmann and Lowey, 1986).