Gel mobility shift assays to detect protein-RNA interactions.
The gel mobility shift assay is a powerful technique for detecting and quantifying protein-RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein-RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein-RNA complexes. In the gel shift assay, protein-RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA buffer, although an alternative Tris-glycine buffering system is superior in many situations. Here, we describe both gel shift methods, along with strategies to improve separation of protein-RNA complexes from free RNA, which can be a particular challenge for small RNA binding proteins.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.
Alexander V
AV
Yakhnin
Helen
H
Yakhnin
Paul
P
Babitzke
D001426:Bacterial Proteins / Q000378:metabolism; D001888:Boric Acids / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005782:Gels; D005998:Glycine / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D001426:Bacterial Proteins; D001888:Boric Acids; C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D005782:Gels; C079092:MtrB protein, Bacteria; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D012313:RNA; D004492:Edetic Acid; C032688:boric acid; D005998:Glycine