FIG. 1.
Immunoblotting of Orp1 and Mcm proteins in soluble and insoluble fractions of fission yeast extract. Whole cell extract (W; lane 1), soluble supernatant (S; lane 2), and insoluble chromatin-enriched pellet (P; lane 3) were prepared from TTY15 cells carrying 5xFLAG-orp1 as described in Materials and Methods. (A) DNA was extracted with phenol, precipitated with ethanol, and analyzed by electrophoresis in a 0.8% agarose gel containing 0.5 μg of ethidium bromide per ml. HindIII-digested λ phage DNA was included as molecular size markers in lane M. (B) Proteins separated by SDS-PAGE were stained with Coomassie brilliant blue. Positions of some molecular size markers are shown on the left. (C) Proteins separated by SDS-PAGE were immunoblotted with anti-FLAG (top), anti-SpMcm6 (middle), and anti-SpMcm2 (bottom) antibodies. Positions of FLAG-Orp1p, SpMcm6p, and SpMcm2p are indicated. Bands migrating faster than SpMcm2p appear to be unrelated proteins, since their abundance did not increase with overproduction of SpMcm2p under the control of the nmt1 promoter (48).