Figure 5

Chemical crosslinking of Mcm4/6/7. Crosslinking of Mcm4/6/7 with glutaraldehyde in the presence and absence of ATP, ADP or ATPγS is shown. A) The samples were analyzed on 3.5% SDS-phosphate gels stained with Coomassie Brilliant Blue ("CB stain"). Each lane contains 9 μg of Mcm4/6/7 in the presence and absence of glutaraldehyde and/or 1 mM ATP, ADP or ATPγS (as indicated). The migration of size standards is indicated on the right and of Mcm4, Mcm6 and Mcm7 in the absence of crosslinker is indicated on the left as are the migration of crosslinked products (by Roman numerals). B) Mcm4/6/7 was crosslinked with glutaraldehyde with and without 5 mM ATP before analysis on a Superose 6 10/300 GL column in the absence of added nucleotide. The A₂₈₀ curves from the columns are shown for Mcm4/6/7 crosslinked in the presence of ATP (black line) and in the absence of ATP (grey line). The peak elution volumes of size standards, analyzed separately are shown above the graph. C) Phosphorimager scan of a SDS-phosphate gel of ³²P-labeled Mcm4^PK/6/7 crosslinked in the presence and absence of ATP (as indicated) is shown. The migrations of crosslinked products, based on Coomassie Blue staining of the same gel are indicated. Western blots to detect Mcm6 (D) and Mcm7 (E) in the crosslinked products. The migrations of Mcm6 and Mcm7 in the absence of crosslinker as well as the migration of crosslinked products are indicated. The presence of glutaraldehyde and ATP are indicated at the top of the blots. Standards with Mcm6 alone (40 ng) or Mcm7 alone (20 ng) are also indicated. The asterisk indicates a cross-reacting band with Mcm7 antibody that is barely visible in the Coomassie stained gels (see A).