title: Isolation of an mRNA-binding protein involved in C-to-U editing.
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.
authors.affiliation: Lerner Research Institute, Department of Cell Biology, The Cleveland Clinic Foundation, Cleveland, OH, USA.
authors.forename: Carri A
authors.initials: CA
authors.lastname: Gerber
authors.forename: Anne
authors.initials: A
authors.lastname: Relich
authors.forename: Donna M
authors.initials: DM
authors.lastname: Driscoll
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003596:Cytosine; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D017393:RNA Editing / Q000235:genetics*; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic; D014498:Uracil
chemical_list: D002352:Carrier Proteins; D012333:RNA, Messenger; D014498:Uracil; D003596:Cytosine; D004247:DNA