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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Peptide tags for labeling membrane proteins in live cells with multiple fluorophores.
TL;DR: This strategy for epitope tagging provides a useful adjunct to green fluorescent protein (GFP)-tagging, which fails to distinguish intracellular from extracellular pools, sometimes interferes with protein localization or function, and requires a separate construct for each color.
Journal ArticleDOI
NS3 helicase from the hepatitis C virus can function as a monomer or oligomer depending on enzyme and substrate concentrations.
Thomas A. Jennings,Samuel G. Mackintosh,Melody K. Harrison,Deniz Sikora,Bartek Sikora,Bhuvanesh Dave,Alan J. Tackett,Craig E. Cameron,Kevin D. Raney +8 more
TL;DR: In this paper, the DNA binding activity of individual subunits within NS3 oligomers was evaluated by labeling two oligonucleotides with fluorescent donor or acceptor molecules and then titrated with NS3.
Journal ArticleDOI
Fluorogen-based reporters for fluorescence imaging: a review.
TL;DR: This review focuses on the recent development of genetically encodable fluorescent reporters that bind endogenously present or exogenously applied fluorogenic chromophores and activate their fluorescence.
Journal ArticleDOI
Effects of As(III) Binding on β-Hairpin Structure
TL;DR: Pairs of cysteines were introduced into a model monomeric β-hairpin to yield a family of peptides such that coordination occurs either across the strands or within the same strand of the β- hairpin to gain an understanding of how arsenic binding influences β-structure.
Journal ArticleDOI
A novel method of affinity-purifying proteins using a bis-arsenical fluorescein.
TL;DR: A novel affinity matrix based on the bis‐arsenical fluorescein dye FlAsH, which specifically recognizes short α‐helical peptides containing the sequence CCXXCC is designed, and it is found that kinesin tagged with this cysteine‐containing helix binds specifically to FlAs H resin and can be eluted in a fully active form.
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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