( fractionation. SYNTH...)Whole Gel Eluter Purification of a Functional Multiprotein DNA Replication Complex, Rev A

SYNTHESOME PURIFICATION PROCEDURES
The DNA synthesome was purified prior to the Whole Gel Eluter step essentially as described by Malkas et al.² and as outlined in Figure 1.

NATIVE PREPARATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS AND ELECTRO-ELUTION
Four percent native polyacrylamide gels, 1.5 mm thick, and containing a 3.5% stacking gel were prepared with a 1.5 mm preparative comb using the Mini-PROTEAN II gel apparatus (Bio-Rad). Sodium dodecyl sulfate (SDS) was excluded from these gels, as well as from the running and sample buffers. Five milligrams of the synthesome protein fraction, purified as described in Figure 1, was loaded onto the gel. Electrophoresis was initially started at 50 volts until the dye front entered the 4% separating PAGE gel, at which time the voltage was increased to 90 volts. Electrophoresis was continued until the dye front reached the bottom of the gel. Following electrophoresis, the PAGE gel was trimmed to fit onto the Mini Whole Gel Eluter as described in the Whole Gel Eluter instruction manual. The gel was then soaked in 20 mM HEPES, pH 7.5, for 10 minutes and layered onto the elution chamber core. The Whole Gel Eluter was assembled as described in the instruction manual, and elution of the resolved synthesome protein fraction from the 4% PAGE gel was carried out as described in the protocol provided with the apparatus. Twenty millimolar HEPES, pH 7.5, was used as the elution buffer in these procedures. The electro-elution was initiated at 60 mA for 1 hour, and then continued at 30 mA for an additional 2 hours. The proteins bound to the cellophane membrane at the end of elution were remo
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