Microtiter plate quantification of mutant and wild-type huntingtin normalized to cell count.
Huntington's disease is caused by a gain-of-function neurotoxic mutation in normally neuroprotective huntingtin. Sensitive assays are required to discriminate mutant huntingtin from wild-type huntingtin. We have developed a normalized 384-plate assay for determination of mutant and wild-type huntingtin. Based on a single pipetting step, the sensitive assay uses two antibody pairs for simultaneous mutant and wild-type huntingtin time-resolved fluorescence resonance energy transfer detection combined with PicoGreen quantification of double-stranded DNA. The assay can be used for discovery of drugs reducing mutant huntingtin over wild-type huntingtin and for assessing the value of huntingtin as a disease progression marker, and it is adaptable to other proteins of interest.
Neuroscience Discovery, Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland. andreas-1.weiss@novartis.com
Andreas
A
Weiss
Stephan
S
Grueninger
Dorothée
D
Abramowski
Francesco Paolo Di
FP
Giorgio
Miriam Moscovitch
MM
Lopatin
H Diana
HD
Rosas
Steven
S
Hersch
Paolo
P
Paganetti
D000906:Antibodies / Q000032:analysis*; D002452:Cell Count; D002460:Cell Line; D004247:DNA; D005347:Fibroblasts / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics; D050505:Mutant Proteins / Q000032:analysis* / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000737:chemistry*; D009687:Nuclear Proteins / Q000032:analysis* / Q000737:chemistry*; D009930:Organic Chemicals
D000906:Antibodies; C086055:HTT protein, human; D000071058:Huntingtin Protein; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA