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
In-cell covalent labeling of reactive His-tag fused proteins
TL;DR: A new method for in-cell protein labeling was developed that employed a binding-induced nucleophilic reaction between the Cys-appended His-tag and the Ni(II)-NTA containing an α-chloroacetamide.
Journal ArticleDOI
Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applications.
Shinya Tsukiji,Itaru Hamachi +1 more
TL;DR: The basic properties of the LDT chemistry and its applications toward in situ engineering and analysis of native proteins in living systems are summarized and current limitations and future challenges are described.
Journal ArticleDOI
Context surrounding processing sites is crucial in determining cleavage rate of a subset of processing sites in HIV-1 Gag and Gag-Pro-Pol polyprotein precursors by viral protease.
TL;DR: It is suggested that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.
Journal ArticleDOI
Speciation of arsenic – A review of phenylarsenicals and related arsenic metabolites
Qingqing Liu,Xiufen Lu,Hanyong Peng,Aleksandra Popowich,Jeffrey Tao,Jagdeesh S. Uppal,Xiaowen Yan,Dana Boe,X. Chris Le +8 more
TL;DR: A review of recent analytical advances in speciation of phenylarsenicals can be found in this article, where the authors discuss the use of high performance liquid chromatography (HPLC) separation with inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectraetry (ESI-MS/MS) detection.
Journal ArticleDOI
Live-cell fluorescent biosensors for activated signaling proteins.
Klaus M. Hahn,Alexei Toutchkine +1 more
TL;DR: A new generation of live-cell fluorescent biosensors enables us to go beyond visualization of protein movements, to quantify the dynamics of many different protein activities, revealing how the location and subtle timing of protein activity controls cell behavior.
References
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Journal ArticleDOI
The green fluorescent protein
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Journal ArticleDOI
Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.
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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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