Fluorescence-based electrophoretic mobility shift assay in the analysis of DNA-binding proteins.
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.
Steiner
Sebastian
S
Institut für Allgemeine Botanik und Pflanzenphysiologie, Lehrstuhl Pflanzenphysiologie Friedrich-Schiller-Universität Jena, Dornburger Str. 159, 07743, Jena, Germany.
Pfannschmidt
Thomas
T
D002846:Chromatography, Affinity; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism
D015342:DNA Probes; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D010940:Plant Proteins; D014157:Transcription Factors