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. 1999 Oct 26;96(22):12367–12372. doi: 10.1073/pnas.96.22.12367

Figure 1.

Figure 1

Glycerol gradient sedimentation of spORC. The concentrated spORC fraction (0.2 ml) eluted from HA antibody cross-linked protein A-agarose beads (as described in Materials and Methods) was loaded onto a 5-ml, 15–35% glycerol gradient containing 0.3 M NaCl in buffer H and centrifuged at 460,000 × g for 14 hr at 4°C; protein standards were centrifuged in parallel gradients. Gradient fractions (20 μl) were subjected to 10% SDS/PAGE separation and immunoblotted with antibodies against spOrc1, 2, and 5 proteins (A) or silver-stained (B). The position of the Orc subunits and the protein bands present in the peak (fraction 11) that were analyzed by peptide sequencing are indicated by asterisks (C). Arrows indicate the position of molecular mass standards: TH, thyroglobulin (669 kDa); CA, catalase (232 kDa); BSA (66 kDa). Lanes: M, molecular mass markers; LO, load on; 1–21, glycerol gradient fraction numbers.