High-throughput protein-DNA measurement using fluorescence anisotropy

Dmitry B. Veprintsev MRC Laboratory of Molecular Biology 09/2008

The tumor suppressor p53 is a transcription factor whose alteration is implicated in 50 % of human cancers. The protein's recognition site contains cytosine bases which may be epigenetically modified with methyl or hydroxymethyl groups, for instance.


The binding affinity of p53 to non-modified and modified sequences was measured using a fluorescently labelled, unmodified reference oligonucleotide. Interaction of protein and oligonucleotide results in high anisotropy. If a modified oligonucleotide displaces the reference molecule, the free fluorescent labeled reference is tumbling faster leading to a lower anisotropy.


The PHERAstar® from BMG LABTECH, interfaced with a liquid handling instrument, provides a powerful multiplexing platform for high throughput determination of binding affinities using fluorescence anisotropy. The sensitivity of the PHERAstar in the fluorescence polarisation mode allowed us to accurately determine of Kd.

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