title: High-throughput quantification of posttranslational modifications in situ by CA-FLIM.
abstract: Signal transduction is mediated by posttranslational modifications (PTMs) of proteins in complex signaling networks. Quantifying PTM levels of multiple network components in response to a stimulus is therefore the key to understand how their concerted activities give rise to cellular function. We have shown that fluorescence lifetime imaging microscopy (FLIM) on cell arrays (CA-FLIM) provides a method to accurately quantify PTM levels of many proteins in situ. Herein, we describe the detailed protocol for CA-FLIM. Less than 2 days are needed from cell array preparation to data analysis, where the main limiting step is the 24h needed for transfection. After generating a single cell array containing 384 spots, it can be imaged and analyzed in less than 2h.
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany.
authors.forename: Hernán E
authors.initials: HE
authors.lastname: Grecco
authors.forename: Pedro
authors.initials: P
authors.lastname: Roda-Navarro
authors.forename: Sven
authors.initials: S
authors.lastname: Fengler
authors.forename: Philippe I H
authors.initials: PI
authors.lastname: Bastiaens
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D046888:Tissue Array Analysis / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria