To determine if Mcm10 binds to CMG in a stable fashion (Figure 2D), we mixed 40 pmol of FLAG-CMG with 120 pmol Mcm10. The mixture was incubated for 15 min on ice and then spun in a microcentrifuge at 15,000 rpm for 10’ at 4°C. The volume of the protein solution was adjusted to 150 μl with binding buffer (25 mM Hepes, pH 7.5; 10% glycerol; 0.01% Nonidet P-40; 300 mM NaCl) and mixed with 25 μl anti-FLAG M2 magnetic beads (50% suspension; Sigma-Aldrich, St. Louis, MO). The protein-bead mixture was incubated on ice for 1 hr and then the beads were collected with a magnetic separator and the supernatant (containing unbound proteins) was removed. The beads were washed three times with 250 μl binding buffer and bound proteins were eluted by incubating in 62.5 μl of the same buffer supplemented with 0.2 mg/ml 3X FLAG peptide on ice for 30’. The beads were collected with a magnetic separator and eluted proteins were collected and analyzed in an 8% SDS-polyacrylamide gel stained with Denville Blue.