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

al relevance to a wide spectrum of basic and applied sciences. Once isolated and characterized, multiprotein DNA replication complexes may provide insight into the mechanisms involved in regulation of the cell cycle, apoptosis, and carcinogenesis. The data presented in this report show that the Whole Gel Eluter can be successfully used in the purification of an intact and functional DNA synthesome. The ease of use of the Whole Gel Eluter, combined with the excellent recovery of the synthesome from native gels, has saved the laboratory time which we have used more effectively toward the further characterization of the synthesome. We are also currently determining whether the lower molecular complex species identified by native PAGE (Figure 2, lanes 24) represent either subassemblies of the synthesome or denaturation products of the replication complex.

The Whole Gel Eluter can also be envisioned to be of potential use in the isolation of transcription and protein synthesis complexes resolved by native PAGE. From our own experience with the apparatus, we found that both the type of elution buffer and the buffer pH are critical features for the elution of a functioning protein complex. We found it necessary to switch from a Tris-based elution buffer to a HEPES-based system in order to significantly increase our recovery of a functional synthesome complex. However, despite the time and effort required to optimize the conditions needed to electro-elute the synthesome from native PAGE using the Whole Gel Eluter, this laboratory feels that the apparatus has greatly facilitated the isolation of a highly purified form of the DNA synthesome, and we have now incorporated the use of the apparatus as part of our standa
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