Journal
ANALYTICAL BIOCHEMISTRY
Volume 411, Issue 1, Pages 155-157Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.12.001
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Funding
- Biotechnology and Biological Sciences Research Council [BB/E005942/1] Funding Source: Medline
- BBSRC [BB/E005942/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/E005942/1] Funding Source: researchfish
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Chemical denaturation of ligand-protein complexes can provide the basis of a label-free binding assay. Here, we show how the technique can be used as a sensitive/affordable screen of potential ligands from a pool of lead drug variants. To demonstrate, we characterized the binding of polyketide ligands based on the mTOR inhibitor rapamycin to the cellular immunophilin FKBP12. This used the intrinsic fluorescence of the protein to monitor the chemical denaturation of each FKBP12-ligand complex. The assay was then successfully modified to a 96-well plate-based screen. Both formats were able to differentiate binding affinities across a wide dynamic range. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.
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