Fluorescence-based electrophoretic mobility shift assay in the analysis of DNA-binding proteins

authored by: Sebastian Steiner, Thomas Pfannschmidt
Abstract: Changes in gene expression mediated by DNA-binding protein factors are a crucial part of many signal transduction pathways. Generally, these regulatory proteins are low abundant and thus their purification and characterisation is labour- and time-intensive. Here we describe a workflow for purification, characterisation and identification of DNA-binding proteins. We show the use of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and describe its advantages for a rapid and convenient screening for regulatory cis-elements. This involves a crude enrichment of nucleic acid binding proteins by heparin-Sepharose chromatography and the characterisation of fractions using overlapping fluorescence-labelled DNA probes spanning the promoter region of interest. The determined protein-binding sites can then be used for sequence-specific DNA-affinity chromatography to purify specifically interacting proteins. Finally, the DNA-binding complexes can be characterised and identified using two-dimensional EMSA, UV-cross-linking and mass spectrometry.
Organisation(s): Institute of Botany
External Organisation(s): Friedrich Schiller University Jena
Type: Article
Journal: Methods in molecular biology (Clifton, N.J.)
Volume: 479
Pages: 273-89
No. of pages: 17
ISSN: 1064-3745
Publication date: 2009
Publication status: Published
Peer reviewed: Yes
Electronic version(s): https://doi.org/10.1007/978-1-59745-289-2_18 (Access: Unknown)

BibTeX

@article{4a0ecccf4874453e917c20238d057436,
title = "Fluorescence-based electrophoretic mobility shift assay in the analysis of DNA-binding proteins",
abstract = "Changes in gene expression mediated by DNA-binding protein factors are a crucial part of many signal transduction pathways. Generally, these regulatory proteins are low abundant and thus their purification and characterisation is labour- and time-intensive. Here we describe a workflow for purification, characterisation and identification of DNA-binding proteins. We show the use of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and describe its advantages for a rapid and convenient screening for regulatory cis-elements. This involves a crude enrichment of nucleic acid binding proteins by heparin-Sepharose chromatography and the characterisation of fractions using overlapping fluorescence-labelled DNA probes spanning the promoter region of interest. The determined protein-binding sites can then be used for sequence-specific DNA-affinity chromatography to purify specifically interacting proteins. Finally, the DNA-binding complexes can be characterised and identified using two-dimensional EMSA, UV-cross-linking and mass spectrometry.",
keywords = "Chromatography, Affinity, DNA Probes/chemistry, DNA-Binding Proteins/analysis, Electrophoretic Mobility Shift Assay/methods, Fluorescent Dyes/chemistry, Plant Proteins/analysis, Protein Binding, Reproducibility of Results, Transcription Factors/analysis",
author = "Sebastian Steiner and Thomas Pfannschmidt",
year = "2009",
doi = "10.1007/978-1-59745-289-2_18",
language = "English",
volume = "479",
pages = "273--89",
journal = "Methods in molecular biology (Clifton, N.J.)",
issn = "1064-3745",
publisher = "Humana Press",
}