To determine if Mcm10 binds to CMG in a stable fashion, we mixed a 3-fold molar excess of Mcm10 (as monomer) with FLAG-tagged CMG and then isolated the CMG-Mcm10 complex using FLAG antibody magnetic beads (Figure 2D). The reaction was washed twice with buffer containing 300 mM NaCl and CMG-Mcm10 complex was eluted using FLAG peptide. The eluted material was analyzed by SDS/PAGE, and stoichiometric levels of Mcm10 were clearly visible with the Mcm2-7 subunits of CMG (lane 3). The GINS subunits of the CMG-Mcm10 complex ran off the gel, but a previous study demonstrated that Mcm10 preferentially associates with the entire CMG complex (Lõoke et al., 2017). This is supported by densitometry analysis of gels showing GINS subunits in both FLAG and MonoQ purified CMG-Mcm10 complexes (Figure 2—figure supplements 3 and 4). Mcm10 was not visible in a control reaction in which Mcm10 was present with beads in the absence of CMG (Figure 2D, lane 2). We also found that the CMG-Mcm10 complex could be isolated using a MonoQ column and elutes at ~400 mM NaCl (Figure 2—figure supplement 4); densitometric scans are consistent with 1–2 Mcm10 per 1 of each CMG subunit (see legend to Figure 2—figure supplement 4 for details). The reconstituted complex is functional in unwinding assays and shows much greater activity on the forked substrate than CMG alone (Figure 2—figure supplement 5), comparable to that seen when adding Mcm10 directly to the unwinding assay in Figure 2. The fact that Mcm10 associates with CMG sufficiently tightly to be isolated by MonoQ chromatography that elutes at 400 mM NaCl and by immunoaffinity beads using 300 mM salt washes suggests that CMG-Mcm10 is a stable complex. Densitometric scans from several preparations of CMG-Mcm10 made using both methods give a stoichiometry of ~1–2 Mcm10 subunits for each CMG complex, consistent with the Mcm10 titration results of Figure 2A.