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Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes
Published on: July 28, 2017
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Horizontal Agarose Gel Mobility Shift Assay for Protein-RNA Complexes.
Jennifer A Ream1, L Kevin Lewis1, Karen A Lewis2
1Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 15, 2018
Summary
This study introduces a fast agarose gel electrophoresis method to measure protein-RNA binding affinity. The accessible native gel mobility shift assay is ideal for routine molecular biology labs.
Area of Science:
- Molecular Biology
- Biochemistry
- Biophysics
Background:
- Agarose gel electrophoresis protocols have advanced for high-resolution DNA and RNA separation.
- Higher voltages and shorter run times enable rapid molecular separations.
Purpose of the Study:
- To develop and describe a protocol for measuring protein-RNA binding affinity using native agarose gel electrophoresis.
- To demonstrate the utility of this assay for characterizing binding constants.
Main Methods:
- Native agarose gel electrophoresis under high-voltage, short-run-time conditions.
- Utilizing a protein-RNA binding system (p19 siRNA binding protein and dsRNA ligand) as a model.
- Optimizing conditions for measuring apparent binding constants.
Main Results:
- Developed a protocol for native agarose gel electrophoresis to assess protein-RNA binding affinity.
- Demonstrated that short electrophoresis times (less than 10 minutes) minimize protein-ligand dissociation.
- Showcased strategies for identifying optimal conditions to determine binding constants.
Conclusions:
- Native agarose gel mobility shift assay is a highly accessible method for characterizing protein-RNA binding affinity.
- The protocol is suitable for common molecular biology laboratories.
- This technique provides a rapid and reliable way to measure binding constants.
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