( on polyethylene glycol precipitation ...)Whole Gel Eluter Purification of a Functional Multiprotein DNA Replication Complex, Rev A

on, polyethylene glycol precipitation, ion-exchange chromatography, and density gradient sedimentation. The integrity of the DNA synthesome has been shown to be maintained after treatment with detergents, salt, RNase, DNase, chromatography on DEAEcellulose (Whatman) and Q-Sepharose (Pharmacia), and following sedimentation in sucrose and glycerol density gradients, indicating that the ready co-purification of the proteins with one another was independent of nonspecific interaction with other cellular macromolecular components.²⁴ Native polyacrylamide gel electrophoresis (PAGE) of the synthesome from HeLa cells revealed the presence of several high molecular weight multiprotein species.⁷ One of these complexes was readily recognized in western blot analysis by a monoclonal antibody against the DNA replication essential protein DNA polymerase α.⁷ This DNA polymerase α containing complex was shown to have a high specific in vitro simian virus 40 (SV40) origin dependent DNA replication activity. We have recently found that Bio-Rads Whole Gel Eluter can greatly aid in the purification of the DNA synthesome. What follows is the description of the conditions we developed for the isolation of the DNA synthesome using the Whole Gel Eluter.

Materials and Methods
CELL CULTURE
Suspension cultures of HeLa cells were grown in Jokliks modified Eagles medium supplemented with 5% each of calf and fetal bovine serum. Exponentially growing cells were harvested and washed three times with phosphate-buffered saline (PBS). The cells were then pelleted by low-speed centrifugation. The cell pellets were stored at -80 C prior to initiating subcellular
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