POL Scientific / JBM / Volume 3 / Issue 1 / DOI: 10.14440/jbm.2016.98
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Removing nucleic acids from nucleoid-Associated proteins purified by affinity column

Audrey Vingadassalon1,2 Philippe Bouloc2 Sylvie Rimsky1
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1 LBPA, ENS Cachan, CNRS, Université Paris-Saclay, F-94235 Cachan, France
2 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris‐Sud, Université Paris‐Saclay, F-91198, Gif‐sur‐Yvette cedex, France
Published: 30 January 2016
© 2016 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

In bacteria, DNA is tightly compacted in a supercoiled organization, which is mediated in part by nucleoid-associated proteins (NAPs). NAPs are well characterized for their ability to bind nucleic acids and for their involvement in gene regulation. A method commonly used to study protein-nucleic acid interactions involves immunoprecipitation of the protein of interest which is subsequently incubated with nucleic acids. A common cause of artifact is due to nucleic acids that remains bound to the protein of interest during the whole purification process. We developed an optimized method for the purification of tagged NAPs on affinity columns. The combination of three known methods allows removal of most of the nucleic acids bound to proteins during the purification process. This protocol is designed to improve the quality and specificity of results of in vitro experiments involving nucleic acid binding tests on purified NAPs. It can be used for in vitro studies of other RNA/DNA binding proteins.

Keywords
affinity
nucleic acids
nucleoid-associated proteins
purification
References

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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific