Document Detail


Fluorogen-activating single-chain antibodies for imaging cell surface proteins.
MedLine Citation:
PMID:  18157118     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.
Authors:
Christopher Szent-Gyorgyi; Brigitte F Schmidt; Brigitte A Schmidt; Yehuda Creeger; Gregory W Fisher; Kelly L Zakel; Sally Adler; James A J Fitzpatrick; Carol A Woolford; Qi Yan; Kalin V Vasilev; Peter B Berget; Marcel P Bruchez; Jonathan W Jarvik; Alan Waggoner
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2007-12-23
Journal Detail:
Title:  Nature biotechnology     Volume:  26     ISSN:  1546-1696     ISO Abbreviation:  Nat. Biotechnol.     Publication Date:  2008 Feb 
Date Detail:
Created Date:  2008-02-08     Completed Date:  2008-03-03     Revised Date:  2008-05-07    
Medline Journal Info:
Nlm Unique ID:  9604648     Medline TA:  Nat Biotechnol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  235-40     Citation Subset:  IM    
Affiliation:
Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA. css@andrew.cmu.edu
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MeSH Terms
Descriptor/Qualifier:
Antibodies, Monoclonal*
Fluorescent Dyes*
Genes, Reporter*
Immunoglobulin Fragments
Membrane Proteins / metabolism*,  ultrastructure
Microscopy, Fluorescence / methods*
Molecular Probe Techniques*
Grant Support
ID/Acronym/Agency:
7 U54 RR022241/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies, Monoclonal; 0/Fluorescent Dyes; 0/Immunoglobulin Fragments; 0/Membrane Proteins
Comments/Corrections
Erratum In:
Nat Biotechnol. 2008 Apr;26(4):470
Note: Schmidt, Brigitte A [corrected to Schmidt, Brigitte F]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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