Fig. 2. Time-courses of the chromatin binding of Cut5 and other replication proteins. (A) Specificity of anti-Cut5 antibody. S-phase egg extracts (1.5 µl) and 125 ng of recombinant Xenopus Cut5 protein (rec.Cut5) were resolved with SDS–PAGE and immunoblotted with anti-Xenopus Cut5 antibody. (B) Sperm nuclei were incubated in 50 µl of S-phase egg extract for the times indicated at 23°C. Chromatin fractions were isolated by centrifugation through a 10% sucrose layer and the isolated chromatin fractions and 1.5 µl of the extracts were resolved by SDS–PAGE, then immunoblotted with antibodies against the various replication proteins indicated.