Nucleosomal organization of macroH2A. (A) Mononucleosomes (red box) were purified using high-density sucrose gradient ultracentrifugation of samples synchronized as in Fig. 1 A. (B) Western blotting before and after immunoprecipitation of pure mononucleosomes using anti-SNAP antibody or rabbit IgG as a negative control. Input samples (and 1:10 dilutions) were loaded in the four lanes on the left. The lower Western blot shows that histone H3 is present in the isolated mononucleosomes and that the loading of the pairs of samples before (pre-S) or after S phase (post-S) lanes is balanced. In the upper blot, there is no evidence for nucleosomes containing SNAP-macroH2A also containing detectable levels of endogenous macroH2A, as the signal intensities in the anti-SNAP lanes do not exceed those of the nonspecific IgG lanes. (C) The brightness of individual nucleosomes containing Oregon Green–labeled SNAP-macroH2A measured by FCS was indistinguishable from individual beads with single molecules of SNAP–Oregon Green, demonstrating that individual nucleosomes contain only single molecules of SNAP-macroH2A. The graph shows means and standard deviations from three independent experiments.