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Updated: Aug 13, 2026

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Published on: February 7, 2019
Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors
A G Papavassiliou1, K Bohmann, D Bohmann
1European Molecular Biology Laboratory, Differentiation Programme, Heidelberg, Germany.
Abstract:
Inducible phosphorylation or dephosphorylation of transcription factors is an important mechanism of signal-dependent gene regulation in eukaryotic cells. This paper describes a combination of techniques that can be used to study the effect of this covalent protein modification on the DNA-binding activity of transcription factors. The protein of interest is genetically tagged with oligohistidine to allow rapid purification on nickel-chelate columns after being transiently overexpressed in eukaryotic tissue culture cells. Phosphorylation-dependent DNA-binding activity is determined in a blotting assay including an in situ dephosphorylation step. Studies on the protooncogene-encoded transcription factor c-Jun employing this assay revealed a TPA-inducible protein dephosphorylation event that strongly increases the DNA-binding potential of the protein. Our data confirm the results published recently by others and provide a rapid, efficient, sensitive, and well-controlled experimental system to analyze the phosphorylation-regulated modulations in the DNA-binding activity of transcription factors.
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