Details
| Original language | English |
|---|---|
| Pages (from-to) | 273-89 |
| Number of pages | 17 |
| Journal | Methods in molecular biology (Clifton, N.J.) |
| Volume | 479 |
| Publication status | Published - 2009 |
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
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In: Methods in molecular biology (Clifton, N.J.), Vol. 479, 2009, p. 273-89.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Fluorescence-based electrophoretic mobility shift assay in the analysis of DNA-binding proteins
AU - Steiner, Sebastian
AU - Pfannschmidt, Thomas
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
KW - Chromatography, Affinity
KW - DNA Probes/chemistry
KW - DNA-Binding Proteins/analysis
KW - Electrophoretic Mobility Shift Assay/methods
KW - Fluorescent Dyes/chemistry
KW - Plant Proteins/analysis
KW - Protein Binding
KW - Reproducibility of Results
KW - Transcription Factors/analysis
U2 - 10.1007/978-1-59745-289-2_18
DO - 10.1007/978-1-59745-289-2_18
M3 - Article
C2 - 19083181
VL - 479
SP - 273
EP - 289
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
SN - 1064-3745
ER -