2011
DOI: 10.1016/j.chembiol.2011.03.014
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Measuring In Vivo Protein Half-Life

Abstract: Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Here, we demonstrate how chemical labeling of SNAP-tag fusion proteins can be exploited to measure the half-life of resident intracellular and extracellular proteins in living mice. First, we demonstrate that SNAP-tag substrates have wide bioavailability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then apply near-infrared probes to perfo… Show more

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Cited by 77 publications
(77 citation statements)
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“…However, this strategy is easily adaptable to other proteins (e.g. Figure 3 D) as well, and similar strategies have been used by us and other investigators, in a range of organisms and for different applications (Jansen et al, 2007;Erhardt et al, 2008;McMurray and Thorner, 2008;Maduzia et al, 2010;Bojkowska et al, 2011;Campos et al, 2011;Dunleavy et al, 2011;Silva et al, in press; also reviewed in O' Hare et al, 2007). We will describe two typical types of SNAP-labeling strategies: Pulse-Chase (Basic Protocol 1) and Quench-Chase-Pulse (Basic Protocol 2), which allow for the analysis of old and new protein pools, respectively.…”
Section: Introductionmentioning
confidence: 82%
“…However, this strategy is easily adaptable to other proteins (e.g. Figure 3 D) as well, and similar strategies have been used by us and other investigators, in a range of organisms and for different applications (Jansen et al, 2007;Erhardt et al, 2008;McMurray and Thorner, 2008;Maduzia et al, 2010;Bojkowska et al, 2011;Campos et al, 2011;Dunleavy et al, 2011;Silva et al, in press; also reviewed in O' Hare et al, 2007). We will describe two typical types of SNAP-labeling strategies: Pulse-Chase (Basic Protocol 1) and Quench-Chase-Pulse (Basic Protocol 2), which allow for the analysis of old and new protein pools, respectively.…”
Section: Introductionmentioning
confidence: 82%
“…BG can be conjugated to a variety of fluorophores and other labels (30), making it possible to tag and track the protein of interest directly by a variety of methods such as fluorescence microscopy, gel electrophoresis, and in vivo imaging (31)(32)(33). The SNAP-tag has been demonstrated not to affect the function of a large number of fusion proteins (34,35) and is an optimal approach for pulse-chase labeling experiments (34,36). The covalent bond between BG and the SNAP-tag, however, makes fluorescence studies of endocytosis difficult because the probe cannot be removed from labeled proteins on the cell surface, concealing the intracellular endocytosed pool.…”
Section: Resultsmentioning
confidence: 99%
“…Pulse-Chase Analysis of SNAP-Tac Cargo Proteins-To analyze the turnover of labeled SNAP-Tac fusion proteins, we designed a degradation assay taking advantage of the covalent labeling of the SNAP-tagged protein by BG (34,37). We labeled cells with BG-800, which allowed us to run total cellular lysates on gels and quantify protein bands directly on an infrared scanner with built-in software to quantify protein bands.…”
Section: Resultsmentioning
confidence: 99%
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“…2 ). Examples of recent applications include single molecule [ 10 , 11 ] and super-resolution imaging [ 12 , 13 ], analysis of protein function [ 14 ], targeted protein inactivation [ 15 ], protein-protein interactions [ 16 , 17 ], protein-drug interactions [ 18 , 19 ], and the determination of protein half-life in animals [ 20 ]. Additionally, similar hAGTbased tag, named CLIP-tag, was developed recently [ 21 ].…”
Section: Introductionmentioning
confidence: 99%