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Isolation of an mRNA-Binding Protein Involved in C-to-U Editing

  • Carri A. Gerber
  • Anne Relich
  • Donna M. Driscoll
Part of the Methods in Molecular Biology book series (MIMB, volume 265)

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.

Key Words

RNA editing RNA-binding protein affinity chromatography Apobec-1 complementation factor 

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Carri A. Gerber
    • 1
  • Anne Relich
    • 1
  • Donna M. Driscoll
    • 1
  1. 1.Lerner Research Institute, Department of Cell BiologyThe Cleveland Clinic FoundationCleveland

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