Development of a method for the high-throughput quantification of cellular proteins.
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.
Paganetti
Paolo
P
Novartis Institutes for Biomedical Research Basel, Novartis Pharma AG, Basel, Switzerland. paolo.paganetti@novartis.com
Weiss
Andreas
A
Trapp
Monique
M
Hammerl
Ina
I
Bleckmann
Dorothée
D
Bodner
Ruth A
RA
Coven-Easter
Shanie
S
Housman
David E
DE
Parker
Christian N
CN
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
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