Abstract
This chapter describes the technique of RNA affinity chromatography, which is a powerful approach for isolating RNA-binding proteins. This method takes advantage of the fact that sequence-specific RNA-binding proteins often bind their targets with high affinity. Here we outline a protocol for purifying Apobec-1 complementation factor (ACF), the RNA-binding subunit of the apolipoprotein-B (apo-B) mRNA-editing enzyme. ACF was purified using synthetic wild-type and mutant apo-B RNAs, which were coupled to cyanogen bromide (CNBr)-activated Sepharose. The methods are plasmid construction for in vitro transcription, affinity chromatography column preparation, protein purification by RNA affinity chromatography, and analysis of the purified protein.
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© 2004 Humana Press Inc.
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Gerber, C.A., Relich, A., Driscoll, D.M. (2004). Isolation of an mRNA-Binding Protein Involved in C-to-U Editing. In: Gott, J.M. (eds) RNA Interference, Editing, and Modification. Methods in Molecular Biology, vol 265. Humana Press. https://doi.org/10.1385/1-59259-775-0:239
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DOI: https://doi.org/10.1385/1-59259-775-0:239
Publisher Name: Humana Press
Print ISBN: 978-1-58829-242-1
Online ISBN: 978-1-59259-775-8
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