Figure 3

Isolation of reconstituted Mcm4/6/7 by size exclusion chromatography. A) A typical elution profile of Mcm4/6/7 from a Superose 6 10/300 GL column. A portion of the indicated fractions was analyzed by Coomassie Brilliant Blue-stained SDS-PAGE (6%). The vertical line marks the border between separate gels. The peak elution of size standards is indicated. The migration of size standards through the SDS gels is indicated on the left and of Mcm4, Mcm6 and Mcm7 on the right. B) Equal volumes of the indicated fractions were analyzed for ATP hydrolysis (white square) or DNA unwinding (black circle) as described in "Methods". C) The absorbance at 280 nm (A₂₈₀) corresponding to the elution of each of Mcm4, Mcm6 and Mcm7 from the gel filtration column are shown. The peaks in absorbance correspond to the peak in the indicated proteins as determined by SDS-PAGE (not shown). The A₂₈₀ curves of size standards (apoferritin, 443 kDa; BioRad standards which include thyroglobulin, 670 kDa; bovine gamma-globulin, 158 kDa and chicken ovalbumin, 44 kDa) are also shown.