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Overvågning af Ubiquitin-proteasom aktivitet i levende celler ved hjælp af en Degron (DGN)-destabiliseret Green Fluorescent Protein (GFP)-baseret Reporter Protein
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Overvågning af Ubiquitin-proteasom aktivitet i levende celler ved hjælp af en Degron (DGN)-destabiliseret Green Fluorescent Protein (GFP)-baseret Reporter Protein

Article DOI: 10.3791/3327-v • 10:25 min • November 10th, 2012
November 10th, 2012 •
1, 1, 1, 2, 1
1Molecular and Cell Biology, Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center

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Fremgangsmåde til overvågning Ubiquitin-proteasom aktivitet i levende celler er beskrevet. En degron-destabiliseret GFP-(GFP-DGN) og en stabil GFP-dgnFS fusionsprotein genereres og transduceres ind i cellen under anvendelse af en lentiviral ekspressionsvektor. Denne teknik gør det muligt at generere en stabil GFP-dgn/GFP-dgnFS udtrykkende cellelinie, hvori Ubiquitin-proteasom aktivitet kan let vurderes ved hjælp af epifluorescens eller flowcytometri.

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Cellular Biology molekylærbiologi medicin Biomedical Engineering Virology proteasome aktivitet lentivirale partikler GFP-DGN GFP-dgnFS GFP humane diploide fibroblaster flowcytometri plasmid vektor
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