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Updated: Mar 24, 2026
Novel RNA-Binding Proteins Isolation by the RaPID Methodology
Published on: September 30, 2016
Northwestern Blot Analysis: Detecting RNA-Protein Interaction After Gel Separation of Protein Mixture
Shangbing Zang1, Ren-Jang Lin2,3
1Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA, 91010, USA.
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.
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01:15Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
02:57Southern Blot
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
01:27SDS-PAGE
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...