Document Detail


Two-color labeling of temporally defined protein populations in mammalian cells.
MedLine Citation:
PMID:  18774715     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in fixed cells. Reactive lissamine rhodamine (LR), 7-dimethylaminocoumarin (DMAC), and bodipy-630 (BDPY) dyes were prepared and examined for use in selective dye-labeling of newly synthesized proteins in Rat-1 fibroblasts. The LR and DMAC, but not BDPY, fluorophores were found to enable selective, efficient labeling of subsets of the proteome; cells labeled with Aha and Hpg exhibited fluorescence emission three- to sevenfold more intense than that of control cells treated with Met. We also examined simultaneous and sequential pulse-labeling of cells with Aha and Hpg. After pulse-labeling, cells were treated with reactive LR and DMAC dyes, and labeled cells were imaged by fluorescence microscopy and analyzed by flow cytometry. The results of these studies demonstrate that amino acid labeling can be used to achieve selective two-color imaging of temporally defined protein populations in mammalian cells.
Authors:
Kimberly E Beatty; David A Tirrell
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2008-08-19
Journal Detail:
Title:  Bioorganic & medicinal chemistry letters     Volume:  18     ISSN:  1464-3405     ISO Abbreviation:  Bioorg. Med. Chem. Lett.     Publication Date:  2008 Nov 
Date Detail:
Created Date:  2008-11-10     Completed Date:  2008-12-08     Revised Date:  2014-09-16    
Medline Journal Info:
Nlm Unique ID:  9107377     Medline TA:  Bioorg Med Chem Lett     Country:  England    
Other Details:
Languages:  eng     Pagination:  5995-9     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Azides / chemistry*
Catalysis
Coumarins / chemistry*
Cyclization
Fibroblasts / metabolism
Fluorescent Dyes / chemical synthesis,  chemistry*
Indicators and Reagents
Methionine / chemistry,  metabolism
Microscopy, Fluorescence / methods
Molecular Structure
Proteins / analysis*
Rats
Grant Support
ID/Acronym/Agency:
GM62523/GM/NIGMS NIH HHS; R01 GM062523/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Azides; 0/Coumarins; 0/Fluorescent Dyes; 0/Indicators and Reagents; 0/Proteins; AE28F7PNPL/Methionine
Comments/Corrections

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