Whole Gel Eluter Purification of a Functional Multiprotein DNA Replication Complex, Rev A ( IN VITRO SV40 REPLICATION ASSA...)

Whole Gel Eluter Purification of a Functional Multiprotein DNA Replication Complex, Rev A

IN VITRO SV40 REPLICATION ASSAY
The assay was performed essentially as described in Malkas et al.²

Results
THE WHOLE GEL ELUTER CAN RAPIDLY AND EFFICIENTLY ELECTRO-ELUTE HIGH MOLECULAR WEIGHT PROTEIN COMPLEXES FROM NATIVE PAGE GELS
We have previously found that when the replicationcompetent DNA synthesome protein fraction, purified from human cells as outlined in Figure 1, was subjected to native PAGE, several distinct high molecular weight protein species were observed.⁷ An example of a silver stained native PAGE resolution of the DNA synthesome protein fraction is shown in Figure 2, lane 5. We have recently identified the specific high molecular weight protein band that contains the fully functional DNA synthesome (Tom et al., to be published elsewhere). The high molecular weight DNA synthesome protein band is indicated by an arrow in Figure 2, lane 5. The initial identification of the discrete DNA synthesome protein band in the native PAGE (Tom et al., to be published elsewhere) required the time consuming procedure of first cutting the gel into small pieces that were then each individually placed into dialysis tubing and the synthesome protein subsequently electro-eluted from the gel using standard methods.¹⁰ This tedious process of obtaining highly purified forms of the DNA synthesome from native PAGE has recently been greatly facilitated by the incorporation of the Whole Gel Eluter into the purification protocol.

The DNA synthesome protein fraction was derived using the procedure outlined in Figure 1. The protein fraction was then resolved by native PAGE as described in the Methods. The native gel
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( IN VITRO SV40 REPLICATION ASSA...)Whole Gel Eluter Purification of a Functional Multiprotein DNA Replication Complex, Rev A