Journal ArticleDOI
Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells
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TLDR
This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.Abstract:
Recombinant proteins containing four cysteines at the i , i + 1, i + 4, and i + 5 positions of an α helix were fluorescently labeled in living cells by extracellular administration of 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.read more
Citations
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Journal ArticleDOI
Inhibition of NF-κB Activation by Arsenite through Reaction with a Critical Cysteine in the Activation Loop of IκB Kinase
Pankaj Kapahi,Takayuki Takahashi,Gioacchino Natoli,Stephen R. Adams,Yi Chen,Roger Y. Tsien,Michael Karin +6 more
TL;DR: Overexpression of IKKβ (C179A) protects NF-κB from inhibition by arsenite, indicating that despite the involvement of a large number of distinct gene products in this activation pathway, the critical target for inhibition by arsenic is on the IKK catalytic subunits.
Journal ArticleDOI
Site-specific labeling of proteins with small molecules in live cells.
Irwin Chen,Alice Y. Ting +1 more
TL;DR: The principal bottleneck for the utilization of small-molecule probes in live cells is the shortage of methodologies for targeting them with very high specificity to biological molecules or compartments of interest.
Journal ArticleDOI
Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.
Marta Fernandez-Suarez,Hemanta Baruah,Laura Martínez-Hernández,Kathleen T. Xie,Jeremy M. Baskin,Carolyn R. Bertozzi,Alice Y. Ting +6 more
TL;DR: The technology for covalent, specific tagging of cellular proteins with chemical probes for live cell imaging is reported, and should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.
Journal ArticleDOI
Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature
TL;DR: The rate of ArfGAP1-catalysed GTP hydrolysis in Arf1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle.
Journal ArticleDOI
The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins.
TL;DR: The green fluorescent protein from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues and will further enhance their use in animal tissues and as intracellular biosensors.
References
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Journal ArticleDOI
The green fluorescent protein
TL;DR: In just three years, the green fluorescent protein from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology.
Journal ArticleDOI
Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.
François Denizot,Rita Lang +1 more
TL;DR: The reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays.
Journal ArticleDOI
Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin
Atsushi Miyawaki,Juan Llopis,Roger Heim,J. Michael McCaffery,Joseph A. Adams,Mitsuhiko Ikura,Mitsuhiko Ikura,Roger Y. Tsien +7 more
TL;DR: New fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations are constructed and dubbed ‘cameleons’.
Journal ArticleDOI
Crystal structure of the Aequorea victoria green fluorescent protein.
TL;DR: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products.
Journal ArticleDOI
Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer
Roger Heim,Roger Y. Tsien +1 more
TL;DR: The results demonstrate that the production of more and better GFP variants is possible and worthwhile, and facilitates multicolor imaging of differential gene expression, protein localization or cell fate.
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