a, Left: SDS–PAGE analysis of samples containing ACF alone or ACF with nucleosomes (20 bp linker DNA) that do not possess the cysteine-reactive crosslinker on the H4 tail. Both samples yield two distinct bands corresponding to the Acf1 and Snf2h subunits (180 kDa and 122 kDa, respectively). Additionally, histone bands at low molecular masses are present in the lane for the sample containing nucleosomes. Right: corresponding immunoblot using α-H4 antibody. In the presence of nucleosomes without crosslinker, a single H4 band is visible at ∼11 kDa corresponding to the histone itself. b, Top: incubation of ACF and nucleosomes that contain a crosslinker at the H4 tail yield Acf1-H4 and Snf2h-H4 crosslinking bands. These bands are absent for ACF without addition of nucleosomes (‘−nucleosomes’) or upon addition of nucleosomes without a crosslinker (‘+nucleosomes (−crosslinker)’). Proteolytic degradation of Acf1 gave rise to a fainter band immediately below Acf1. Bottom: α-histone H4 immunoblotting reveals specific Acf1-H4 and Snf2h-H4 bands that are absent for ACF without addition of nucleosomes or upon addition of nucleosomes without a crosslinker.