Out with the old, in with the new? Comparing methods for measuring protein degradation

Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library product...

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Bibliographic Details
Published in:Cell biology international 2011-05, Vol.35 (5), p.457-462
Main Authors: Yewdell, Jonathan W, Lacsina, Joshua R, Rechsteiner, Martin C, Nicchitta, Christopher V
Format: Article
Language:English
Subjects:
Animals
Biochemistry - methods
Cloning, Molecular - methods
Flow Cytometry - methods
GPSP
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Humans
metabolic labelling
protein degradation
Proteins - genetics
Proteins - metabolism
proteolysis
proteostasis
pulse-chase
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
SILAC
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Summary:Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library products by flow cytometry [Yen et al. (2008) Science 322, 918]. While no method is perfect, we conclude that chimeric gene reporter approaches, though powerful, should be applied cautiously, due principally to GFP (or other reporter tag) interference with protein organelle targeting or incorporation into macromolecular assemblies, both of which cause spuriously high degradation rates.
ISSN:1065-6995
1095-8355
DOI:10.1042/CBI20110055