PMC:548366 / 7018-8704 JSONTXT

{"project":"2_test","denotations":[{"id":"15687385-9251047-77096027","span":{"begin":175,"end":177},"obj":"9251047"},{"id":"15687385-7760937-77096028","span":{"begin":528,"end":530},"obj":"7760937"},{"id":"15687385-9251047-77096029","span":{"begin":531,"end":533},"obj":"9251047"},{"id":"15687385-8861912-77096030","span":{"begin":534,"end":536},"obj":"8861912"}],"text":"Purification from Xenopus egg extract\nThe egg extract for purification was prepared after in vivo activation of Xenopus eggs by an addition of A23187 as described previously (31). Fifty percent (w/v) polyethylene glycol 6000 (PEG) (Wako) in 20 mM HEPES-KOH, pH 8 was added to the extract to make 5% PEG to obtain precipitated and supernatant fractions. A partially purified fraction containing Cdc6 and Cdt1 (BPAS fraction) was obtained from the 5% PEG precipitated fraction by a process based on a method described previously (16,31,32). In brief, the precipitated fraction was solubilized in T'LFB1 (40 mM HEPES-KOH, pH 8.0, 20 mM potassium phosphate, 2 mM MgCl2, 1 mM EGTA, 2 mM DTT, 10% (w/v) sucrose, 0.01% (v/v) Triton X-100, 1 μg/ml pepstatin and 1 μg/ml leupeptine) supplemented with 150 mM KCl, and adsorbed onto phosphocellulose resin (P11, Whatman). The adsorbed proteins were eluted with T'LFB1 containing 500 mM KCl. The BPAS fraction was obtained by 40% saturated ammonium sulfate precipitation from the phosphocellulose eluate and was solubilized in T'LFB1. A partially purified Cdc6 fraction was prepared from the BPAS fraction after removal of Cdt1 as indicated in ‘Immunodepletion’.\nMcm2-7 complex was partially purified from the 5% PEG supernatant fraction. The fraction was subjected to further precipitation by 12% PEG, and the 5–12% PEG precipitated fraction was adsorbed by Q-Sepharose FF (Amersham Biosciences) in T'LFB1 containing 100 mM KCl. The adsorbed proteins were eluted with T'LFB1 containing 350 mM KCl, and precipitated with the addition of an equal volume of 50% PEG. The purified fraction was solubilized with T'LFB1 and stored at −80°C prior to use."}