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Quantitative distance information on protein‐DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer
Author(s) -
Lorenz Mike,
Diekmann Stephan
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683()22:6<990::aid-elps990>3.0.co;2-x
Subject(s) - förster resonance energy transfer , polyacrylamide , fluorescence , dna , chemistry , fluorescein , polyacrylamide gel electrophoresis , gel electrophoresis , resonance (particle physics) , biophysics , biochemistry , biology , optics , physics , particle physics , polymer chemistry , enzyme
In polyacrylamide gels, we have quantitatively determined Förster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein‐DNA complexes. The donor‐dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change. The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Förster distance R 0 of 46 Å compared to 50 Å in solution. We have determined global structural properties of two proteins binding to double‐labeled DNA using a novel gel‐based fluorescence resonance energy transfer assay. In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA. The measured Förster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex.