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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Patent
Covalent tethering of functional groups to proteins and substrates therefor
Aldis Darzins,Lance P. Encell,Dieter Klaubert,Georgyi V. Los,Mark McDougall,Keith V. Wood,Monika G. Wood,Chad Zimprich +7 more
TL;DR: In this article, a mutant hydrolase can be fused to a protein of interest, and a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein is provided.
Phospholamban Pentamer Quaternary Conformation Determined by In-Gel
TL;DR: The data support a helical pinwheel model for the PLB pentamer, in which the cytoplasmic domains bend sharply outward from the central bundle of helices, and compare favorably to distances predicted by a computer molecular model of the phospholamban pentamer constructed from NMR solution structures.
Journal ArticleDOI
Extension of the applicable range of fluorescein: a fluorescein-based probe for Western blot analysis.
Mako Kamiya,Yasuteru Urano,Yasuteru Urano,Nobuyoshi Ebata,Masami Yamamoto,Jyunichi Kosuge,Tetsuo Nagano +6 more
Journal ArticleDOI
LDAI-Based Chemical Labeling of Intact Membrane Proteins and Its Pulse-Chase Analysis under Live Cell Conditions
Takayuki Miki,Sho-hei Fujishima,Kazuhiro Komatsu,Keiko Kuwata,Shigeki Kiyonaka,Itaru Hamachi +5 more
TL;DR: It is demonstrated that ligand-directed acyl imidazole (LDAI) chemistry is broadly applicable to selective chemical labeling of various types of membrane-bound proteins under live cell conditions without a need for any tag fragments.
Journal ArticleDOI
Ligand-directed dibromophenyl benzoate chemistry for rapid and selective acylation of intracellular natural proteins
TL;DR: In this article, a ligand-directed dibromophenyl benzoate (LDBB) chemistry was developed for the acylation of proteins in living cells on the basis of liganddirected chemistry.
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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