Abstract
Transcription machinery plays a central role in both the gene expression and nucleoid compaction. In this chapter we elaborate on the optimization of RNA polymerase purification protocol using a mild procedure with the purpose of preserving its native composition. This protocol combines protein extraction under non-denaturing conditions, heparin based affinity purification, and consequent BN-PAGE–SDS-PAGE separation. The outcome is an experimental procedure for screening RNA polymerase composition with associated proteins, in various bacterial strains or mutant backgrounds. With modifications in the column purification step, this procedure can be applied for isolation and identification of the components of other multi-protein complexes.
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (DFG MU 1549/7-1). The authors thank Steffi Teresa Jimmy and Zharko Daniloski (respectively carrying out their MSc and BSc thesis work in the lab) who participated in the development of the method and used it to generate results.
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Mehandziska, S., Petrescu, A.M., Muskhelishvili, G. (2017). Isolation and Analysis of RNA Polymerase Supramolecular Complex with Associated Proteins. In: Espéli, O. (eds) The Bacterial Nucleoid. Methods in Molecular Biology, vol 1624. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7098-8_9
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DOI: https://doi.org/10.1007/978-1-4939-7098-8_9
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Publisher Name: Humana Press, New York, NY
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