Photoaffinity labelling displacement assay using multiple recombinant protein domains

David J. Fallon, Alex Phillipou, Christopher J. Schofield, David House, Nicholas C. O. Tomkinson, Jacob T. Bush

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The development and optimisation of a photoaffinity labelling (PAL) displacement assay is presented, where a highly efficient PAL probe was used to report on the relative binding affinities of compounds to specific binding sites in multiple recombinant protein domains in tandem. The N- and C-terminal bromodomains of BRD4 were used as example target proteins. A test set of 264 compounds annotated with activity against the bromodomain and extra-terminal domain (BET) family in ChEMBL were used to benchmark the assay. The pIC50 values obtained from the assay correlated well with orthogonal TR‑FRET data, highlighting the potential of this highly accessible PAL biochemical screening platform.
Original languageEnglish
Pages (from-to)1183-1197
Number of pages15
JournalBiochemical Journal
Issue number15
Early online date4 Jul 2023
Publication statusPublished - 31 Aug 2023


  • photoaffinity
  • assay
  • bromodomains


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