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Scheme of an mutant huntingtin aggregation assay modified for drug screening

Wang, Jin ; Gines, Silvia ; E MacDonald, Marcy ; F Gusella, James

figshare 2011

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  • Título:
    Scheme of an mutant huntingtin aggregation assay modified for drug screening
  • Autor: Wang, Jin ; Gines, Silvia ; E MacDonald, Marcy ; F Gusella, James
  • Assuntos: Genetics not elsewhere classified
  • Descrição: Copyright information:Taken from "Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation"BMC Neuroscience 2005;6():1-1.Published online 13 Jan 2005PMCID:PMC548150.Copyright © 2005 Wang et al; licensee BioMed Central Ltd. In the primary screening, the mixture of fusion protein, GST-Q58-Htn (20 μg/ml) and Thrombin (0.5 unit/μg protein) was immediately distributed into the 96-well plates containing diluted compounds at 40 μl/well. The final concentration of the small compounds is 100 μM. 10 μl of 10% SDS/50 mM 2-mercaptoethanol was added into each well to stop the reaction after 24 hours incubation at room temperature. The aggregates were separated by filtering through a cellulose acetate membrane (0.2 μm). Immunoblotting was done with a specific anti-huntingtin antibody, HP1, followed by incubation with peroxidase conjugated anti-rabbit antibody. The signals of the retained aggregates were scanned and quantified. In the secondary screening, compounds tested positive in were tested at 10 μM and a 45-minute incubation at room temperature was followed after mixing the protein and enzyme. : A typical immunoblot of the huntingtin aggregation inhibitor screening. The aggregates retained on the membrane were visualized by ECL. The intensity of dot reflects the amount of aggregates. The blot shows the positions of each drug in a 96-well plate. Congo Red: positive control (E, 12 and F, 12). DMSO: negative control (G, 12 and H, 12). Drugs in wells F2, E4, E9 and D3 are huntingtin aggregation inhibitors.
  • Editor: figshare
  • Data de criação/publicação: 2011
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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