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
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Förster resonance energy transfer - A spectroscopic nanoruler: Principle and applications
TL;DR: The basic principles and applications of FRET in chemistry, biology, and physics are discussed and the recent improvements in optical techniques facilitate the measurements of two-dimensional spatial distribution in steady-state as well as dynamic bimolecular interactions.
Journal ArticleDOI
A combined approach to improving large-scale production of tobacco etch virus protease.
Paul G. Blommel,Brian G. Fox +1 more
TL;DR: A quantitative, high-throughput assay for TEV protease activity in cell lysates is used to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods to test whether further improvements are possible.
Journal ArticleDOI
Fluorescence visualization of newly synthesized proteins in mammalian cells.
Kimberly E. Beatty,Julie C. Liu,Fang Xie,Daniela C. Dieterich,Erin M. Schuman,Qian Wang,David A. Tirrell +6 more
TL;DR: It is reported that selective fluorescence labeling and imaging of newly synthesized proteins can be accomplished in a diverse set of mammalian cells.
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Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo
TL;DR: It is demonstrated that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytotic treatment using a functional SyT I protein.
Journal ArticleDOI
Monitoring protein interactions and dynamics with solvatochromic fluorophores
TL;DR: In this article, the authors discuss recent technological advancements and their application in the design of powerful reporters, which serve critical roles in modern cell biology and assay development, and discuss recent developments in site-selective methods to incorporate these chemical tools into proteins.
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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