title: Development of a method for the high-throughput quantification of cellular proteins.
abstract: The quantification of cellular proteins is essential for the study of many different biological processes. This study describes an assay for the detection of the intracellular mutant huntingtin, the causative agent of Huntington's disease, with a method that may be generally applicable to other cellular proteins. A small recombinant protein tag that is recognized by a pair of readily available, high-affinity monoclonal antibodies was designed. This tag was then added to an inducible fragment of the mutant huntingtin protein by genetic engineering. We show that it is possible to use time-resolved FRET to detect low intracellular levels of huntingtin by a simple lysis and detection procedure. This assay was then adapted into a homogeneous, miniaturized format suitable for screening in 1536-well plates. The use of time-resolved FRET also permits the assay to be multiplexed with a standard readout of cell toxicity, thus allowing the identification of conditions causing reduction of protein levels simply due to cytotoxicity. The screening results demonstrated that the assay is able to identify compounds that modulate the levels of huntingtin both positively and negatively and that represent valuable starting points for drug discovery programs.
authors.affiliation: Novartis Institutes for Biomedical Research Basel, Novartis Pharma AG, Basel, Switzerland. paolo.paganetti@novartis.com
authors.forename: Paolo
authors.initials: P
authors.lastname: Paganetti
authors.forename: Andreas
authors.initials: A
authors.lastname: Weiss
authors.forename: Monique
authors.initials: M
authors.lastname: Trapp
authors.forename: Ina
authors.initials: I
authors.lastname: Hammerl
authors.forename: Dorothée
authors.initials: D
authors.lastname: Bleckmann
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Bodner
authors.forename: Shanie
authors.initials: S
authors.lastname: Coven-Easter
authors.forename: David E
authors.initials: DE
authors.lastname: Housman
authors.forename: Christian N
authors.initials: CN
authors.lastname: Parker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D000071058:Huntingtin Protein; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis* / Q000235:genetics; D010078:Oxazines / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D054852:Small Molecule Libraries; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010078:Oxazines; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D014966:Xanthenes; C005843:resazurin