Electrophoretic mobility shift assays with GFP-tagged proteins (GFP-EMSA)

Sorenson, Alanna E., and Schaeffer, Patrick M. (2020) Electrophoretic mobility shift assays with GFP-tagged proteins (GFP-EMSA). In: Labrou, Nikolaos E., (ed.) Targeting Enzymes for Pharmaceutical Development. Methods in Molecular Biology, 2089 . Springer, New York, NY, USA, pp. 159-166.

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Abstract

The electrophoretic mobility shift assay (EMSA) is commonly used for the study of nucleic acid-binding proteins. The technique can be used to demonstrate that a protein is binding to RNA or DNA through visualization of a shift in electrophoretic mobility of the nucleic acid band. A major disadvantage of the EMSA is that it does not always provide an absolute certitude that the band shift is due to the protein under scrutiny, as contaminants in the sample could also cause the band shift. Here we describe a variation of the standard EMSA allowing to visualize with added certitude, the co-localized band shifts of a GFP-tagged protein binding to its cognate nucleic acid target sequence stained with an intercalator, such as GelRed. Herein, we present an illustrative protocol of this useful technique called GFP-EMSA along with specific notes on its advantages and limitations.

Item ID: 65794
Item Type: Book Chapter (Scholarly Work)
ISBN: 978-1-0716-0163-1
ISSN: 1064-3745
Keywords: Green fluorescent protein, Nucleic acid binding, DNA binding, RNA binding, Electro-phoretic mobility shift assay, Band shift assay, Gel shift assay
Copyright Information: © Springer Science+Business Media, LLC 2020
Date Deposited: 15 Mar 2021 02:29
FoR Codes: 31 BIOLOGICAL SCIENCES > 3101 Biochemistry and cell biology > 310109 Proteomics and intermolecular interactions (excl. medical proteomics) @ 100%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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