title: Northwestern Blot Analysis: Detecting RNA-Protein Interaction After Gel Separation of Protein Mixture.
abstract: Northwestern assays detect a direct binding of a given RNA molecule to a protein immobilized on a nitrocellulose membrane. Here, we describe protocols to prepare (32)P-labeled RNA probes and to use them to assay for RNA-protein interactions after partially purified protein preparations are resolved on denaturing SDS-polyacrylamide gels. The method can unambiguously determine whether the protein of interest can directly and independently bind RNA even in the presence of contaminating bacterial proteins or degradation products that at times may hinder interpretation of results obtained from gel mobility shift or RNP immunoprecipitation assays.
authors.affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA, 91010, USA.
authors.forename: Shangbing
authors.initials: S
authors.lastname: Zang
authors.affiliation: Irell & Manella Graduate School of Biological Sciences of the City of Hope, Duarte, CA, USA. RLin@coh.org.|Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA, 91010, USA. RLin@coh.org.
authors.forename: Ren-Jang
authors.initials: RJ
authors.lastname: Lin
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D015152:Blotting, Northern / Q000379:methods*; D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D015347:RNA Probes / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D015347:RNA Probes; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA
keywords: 32P-labeling of RNA; Cobalt affinity purification of histidine-tagged protein; Direct RNA binding; In vitro transcription; Protein renaturation on membrane; RNA-binding protein; SP6 or T7 RNA polymerase