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Author Notes:

miao@ku.edu; bo.liang@emory.edu

Conceptualization, Y.M. and B.L.; investigation, Y.G., D.C., S.P., and S.A.; writing – original draft, Y.G., S.P., Y.M., and B.L.; writing – review & editing, Y.M. and B.L.; funding acquisition, Y.M. and B.L.; supervision, Y.M. and B.L.

We want to thank Dr. Karen Kirby for the MST training and Dr. Stefan Sarafianos for allowing us to use the MST instrument. We thank the Liang group members at Emory University School of Medicine for performing the experimental studies, including protein purification and MST assays, and the members of the Miao group at the University of Kansas for computational simulations.

The authors declare no competing interests.

Subjects:

Research Funding:

B.L. was supported by NIGMS R01GM130950 and the Start-Up Fund from Emory University School of Medicine. Y.M. was supported by startup funding from the College of Liberal Arts and Sciences at the University of Kansas.

Keywords:

  • Bioinformatics
  • Protein biochemistry
  • Protein expression and purification

Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations.

Tools:

Journal Title:

STAR Protoc

Volume:

Volume 2, Number 1

Publisher:

, Pages 100315-100315

Type of Work:

Article | Final Publisher PDF

Abstract:

Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression and purification step, including high salt and heparin column for contaminant RNA removal. After the assembly of RNA into the ribonucleoprotein complex, the MicroScale Thermophoresis assay enables the binding affinity to be obtained quickly with a small amount of sample. Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020).

Copyright information:

© 2021 The Author(s)

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/).
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