Frapid: achieving full automation of FRAP for chemical probe validation.

Fluorescence Recovery After Photobleaching (FRAP) is an established method for validating chemical probes against the chromatin reading bromodomains, but so far requires constant human supervision. Here, we present Frapid, an automated open source code implementation of FRAP that fully handles cell identification through fuzzy logic analysis, drug dispensing with a custom-built fluid handler, image acquisition & analysis, and reporting. We successfully tested Frapid on 3 bromodomains as well as on spindlin1 (SPIN1), a methyl lysine binder, for the first time.

[1]  Alberto Diaspro,et al.  Fuzzy logic and maximum a posteriori-based image restoration for confocal microscopy. , 2006, Optics letters.

[2]  Karsten König,et al.  Software-aided automatic laser optoporation and transfection of cells , 2015, Scientific Reports.

[3]  J. Brodbelt,et al.  Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination , 2015, Genes & development.

[4]  M. Hendzel,et al.  Kdm4b Histone Demethylase Is a DNA Damage Response Protein and Confers a Survival Advantage following γ-Irradiation* , 2013, The Journal of Biological Chemistry.

[5]  S. Knapp,et al.  PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains. , 2013, Cancer research.

[6]  R. Klose,et al.  5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation. , 2013, Chemical science.

[7]  T Misteli,et al.  Protein dynamics: implications for nuclear architecture and gene expression. , 2001, Science.

[8]  A. Hyman,et al.  FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange. , 2010, Biophysical journal.

[9]  C. Rice,et al.  Suppression of inflammation by a synthetic histone mimic , 2010, Nature.

[10]  Gary D Bader,et al.  Pathway and network analysis of cancer genomes , 2015, Nature Methods.

[11]  H. Weinmann,et al.  Bromodomains and Their Pharmacological Inhibitors , 2014, ChemMedChem.

[12]  Raibatak Das,et al.  Analysis of membrane-localized binding kinetics with FRAP , 2008, European Biophysics Journal.

[13]  M. Jones,et al.  Identification and characterization of BPTF, a novel bromodomain transcription factor. , 2000, Genomics.

[14]  William B. Smith,et al.  Selective inhibition of BET bromodomains , 2010, Nature.

[15]  A. Emili,et al.  Discovery of a chemical probe for the L3MBTL3 methyl-lysine reader domain , 2012, Nature chemical biology.

[16]  Stefan Knapp,et al.  Bromodomains as therapeutic targets , 2011, Expert Reviews in Molecular Medicine.

[17]  P. D. de Jong,et al.  Ligation-independent cloning of PCR products (LIC-PCR). , 1990, Nucleic acids research.

[18]  S. Müller,et al.  Epigenetic Chemical Probes , 2012, Clinical pharmacology and therapeutics.

[19]  Jan Ellenberg,et al.  Micropilot: automation of fluorescence microscopy–based imaging for systems biology , 2011, Nature Methods.

[20]  A. Kenworthy,et al.  Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells. , 2005, Methods.

[21]  Clarence Yapp,et al.  Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching , 2014, Journal of biomedical optics.

[22]  Fangxue Sherry He,et al.  Systematic identification of mammalian regulatory motifs' target genes and functions , 2008, Nature Methods.

[23]  A. Gingras,et al.  Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching , 2014, Epigenetics & Chromatin.