Chapter 14
Gel-Based Analysis of Protein–Nucleic Acid Interactions
James A. W. Stowell, Terence T. L. Tang, Maximilian Seidel,
and Lori A. Passmore
Abstract
Electrophoretic mobility shift assays (EMSAs) are among the most frequently used and straightforward
experiments for studying protein–nucleic acid interactions. EMSAs rely on the principle that protein–nucleic acid complexes have reduced electrophoretic mobility in a native gel matrix compared to free nucleic
acid due to their larger size and reduced negative charge. Therefore, bands for the protein–nucleic acid
complexes are shifted in a gel and can be distinguished from free nucleic acids. EMSAs remain a popular
technique since they do not require specialist equipment and the complexes formed are easily visualized.
Furthermore, the technique can be adapted to enable various aspects of protein–nucleic acid interactions to
be investigated, including sequence specificity, estimated binding affinity, and binding stoichiometry.
Key words EMSA, Electrophoretic mobility shift assay, Band shift assay, Protein–DNA interactions,
Protein–RNA interactions, Affinity measurement
1 Introduction
Protein–nucleic acid interactions coordinate many fundamental
cellular processes, including DNA replication, transcription, RNA
processing, and translation. The electrophoretic mobility shift assay
(EMSA), or gel shift assay, is a straightforward but sensitive method
of characterizing protein–nucleic acid interactions. Although
EMSAs are typically used for qualitative purposes, they can provide
quantitative estimates of dissociation constants (affinity measurements), binding stoichiometry, and sequence and structural
specificity [1].
Gel-based detection of protein–nucleic acid complexes was first
described for the DNA-binding lactose operon regulatory network
[2, 3]. The method anticipates that under electrophoretic conditions, larger protein-bound nucleic acids are retarded within a
native gel matrix, whereas unbound nucleic acids have higher electrophoretic mobility [4]. In a vertical electrophoresis apparatus,
free nucleic acid is generally found at the bottom of the gel towards
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
321
Gel-Based Analysis of Protein–Nucleic Acid Interactions
James A. W. Stowell, Terence T. L. Tang, Maximilian Seidel,
and Lori A. Passmore
Abstract
Electrophoretic mobility shift assays (EMSAs) are among the most frequently used and straightforward
experiments for studying protein–nucleic acid interactions. EMSAs rely on the principle that protein–nucleic acid complexes have reduced electrophoretic mobility in a native gel matrix compared to free nucleic
acid due to their larger size and reduced negative charge. Therefore, bands for the protein–nucleic acid
complexes are shifted in a gel and can be distinguished from free nucleic acids. EMSAs remain a popular
technique since they do not require specialist equipment and the complexes formed are easily visualized.
Furthermore, the technique can be adapted to enable various aspects of protein–nucleic acid interactions to
be investigated, including sequence specificity, estimated binding affinity, and binding stoichiometry.
Key words EMSA, Electrophoretic mobility shift assay, Band shift assay, Protein–DNA interactions,
Protein–RNA interactions, Affinity measurement
1 Introduction
Protein–nucleic acid interactions coordinate many fundamental
cellular processes, including DNA replication, transcription, RNA
processing, and translation. The electrophoretic mobility shift assay
(EMSA), or gel shift assay, is a straightforward but sensitive method
of characterizing protein–nucleic acid interactions. Although
EMSAs are typically used for qualitative purposes, they can provide
quantitative estimates of dissociation constants (affinity measurements), binding stoichiometry, and sequence and structural
specificity [1].
Gel-based detection of protein–nucleic acid complexes was first
described for the DNA-binding lactose operon regulatory network
[2, 3]. The method anticipates that under electrophoretic conditions, larger protein-bound nucleic acids are retarded within a
native gel matrix, whereas unbound nucleic acids have higher electrophoretic mobility [4]. In a vertical electrophoresis apparatus,
free nucleic acid is generally found at the bottom of the gel towards
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
321