Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA.
Electrophoretic mobility shift assays (EMSAs) are commonly used to analyze nucleic acid-protein interactions. When nucleic acid is bound by protein, its mobility during gel electrophoresis is reduced. Similarly, the final position of protein within a complex is shifted when compared to its free state. Here we provide a protocol for a simple approach that uses these mobility differences to identify nucleic acid-binding proteins. Following EMSA, denaturing gel electrophoresis is implemented to provide a second dimension of separation. Protein that binds a specific nucleic acid is identified as a spot(s) whose presence at a particular position(s) is dependent on nucleic acid within the initial binding reaction. The polypeptide in a spot can be subsequently identified by mass spectrometry. As EMSAs can be performed using partially purified or cell extracts, this approach substantially reduces the need for protein purification. It should facilitate the identification of a nucleic acid-binding protein within approximately 4 d.
Stead
Jonathan A
JA
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
McDowall
Kenneth J
KJ
D004268:DNA-Binding Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay; D010449:Peptide Mapping; D016601:RNA-Binding Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins