10.7554/eLife.29118.007Figure 2—figure supplement 4.MonoQ reconstitution of CMG/Mcm10 complex.To form a CMG-Mcm10 complex, 600 μg purified CMG (765 pmol) was mixed with 250 μg purified Mcm10 (3.1 nmol). The mixture was incubated on ice for 30’ and spun in a microcentrifuge at 15,000 rpm for 3’ at 4°C to remove any precipitated proteins. The CMG/Mcm10 mixture was injected at 0.1 ml/min onto a 100 μl monoQ column that had previously been equilibrated in a buffer consisting of 20 mM Tris-Cl pH7.5, 200 mM KCl, 2 mM DTT and 2 mM MgCl2. The column was washed and then eluted in the same buffer using a 2.5 ml gradient from 200 mM to 600 mM KCl, collecting 0.1 ml fractions. Peak fractions were analyzed in an 8% SDS-polyacrylamide gel stained with Denville Blue. A densitometric scan of the gel showed a molar ratio of 1.0 Mcm2-7: 1.59 Cdc45: 2.0 Mcm10: 1.4 Sld5: Psf1 and Psf2 at 1.05 each (Psf1/2 co-migrate, and together give a stoichiometry of 1.0:2.1 for Mcm2-7: Psf1 +Psf2); Psf3 runs at the dye front.