Direct pH Measurements by using Subcellular Targeting of 5(and 6-) Carboxyseminaphthorhodafluor in Mammalian Cells

Benink, Hélène A; McDougall, Mark G.; Klaubert, Dieter H.; Los, Georgyi V.

doi:10.2144/000113220

Cited by 29 publications

(28 citation statements)

References 26 publications

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“…Chemical tags have been used to study the localization and dynamics of proteins in living cells, especially in experiments that can not be easily performed with fluorescent proteins. In the last few years, chemical tags have been used to label proteins with fluorophores suited for advanced imaging technologies such as super-resolution (SR) microscopy [57][58][59], Ca 2þ -imaging [60][61][62], pH sensing [63], hydrogen peroxide detection [64], chromophore assisted light inactivation [36,65,66], and multi-photon microscopy [19]. Recently, Kosaka et al demonstrated the use of the Halo-tag to perform in vivo imaging studies in live animals for the first time [67].…”

Section: Applications Of Chemical Tags In Live Cell Imagingmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Section: Applications Of Chemical Tags In Live Cell Imagingmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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“…The SNAP-tag technology has recently been applied to targeting sensors for Zn 2+ [63], Ca 2+ [64][65][66], and probes for hydrogen peroxide [67]. This strategy has also been applied to pH sensors that can be covalently linked to HaloTag [68]. A recent example for the nuclear localization of a highly sensitive BODIPY-based Ca 2+ -sensitive dye is shown in Figure 2b [66].…”

Section: Applications Of Tag-mediated Labeling Study Of Protein Functmentioning

confidence: 99%

How to obtain labeled proteins and what to do with them

Hinner

Johnsson

2010

Current Opinion in Biotechnology

259

217

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We review new and established methods for the chemical modification of proteins in living cells and highlight recent applications. The review focuses on tag-mediated protein labeling methods, such as the tetracysteine tag and SNAP-tag, and new developments in this field such as intracellular labeling with lipoic acid ligase. Recent promising advances in the incorporation of unnatural amino acids into proteins are also briefly discussed. We describe new tools using tag-mediated labeling methods including the super-resolution microscopy of tagged proteins, the study of the interactions of proteins and protein domains, the subcellular targeting of synthetic ion sensors, and the generation of new semisynthetic metabolite sensors. We conclude with a view on necessary future developments, with one example being the selective labeling of non-tagged, native proteins in complex protein mixtures. IntroductionChemists are becoming increasingly fascinated with derivatizing proteins by genetically non-encodable synthetic molecules. As discussed in this review, such molecules include fluorescent dyes, chemical crosslinkers, pharmacologically active compounds, and synthetic fluorescent probes for ions such as Ca 2+ . The labeling of proteins with synthetic molecules provides exciting new tools for studying proteins and their function in the cell, and is promising to have a strong impact on drug development. In this review, we will provide a concise overview over established and new methods that provide proteins derivatized with a label, and give illustrative recent examples of their application. For further information, the reader is directed to recently published more exhaustive reviews [1][2][3][4]. The focus of our review lies on covalent tag-based labeling methods, as these allow an efficient and irreversible transfer of labels in mammalian cells. However, we will also comment on unnatural amino acid incorporation as a tool of increasing importance for mammalian cell studies. The discussion of methods and applications is preceded by general considerations on tag-mediated labeling. We will conclude with an outlook on future directions and highlight areas that would profit most from new developments. Tag-mediated labelingAn ideal method for tag-based protein labeling should exhibit the following features: (i) the possibility to introduce any label of choice in one step, (ii) fast and quantitative labeling, (iii) no labeling of non-target proteins, (iv) a small tag to minimize its impact on protein function, (v) the formation of a stable, covalent bond between protein and label, and (vi) no side effects of the reagents used for labeling. Finally, an ideal tag should work in vitro, in complex protein samples, on the cell surface, within the cell and cellular compartments, and in living animals (in vivo), with this order representing an increasing level of difficulty. None of the existing labeling methods fulfils all these requirements. It is notable, however, that the existing methods of tag-mediated labeling can be grouped ...

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“…Covalent linking of an environmentally sensitive dye to a protein of interest, allows local interrogation of the chemical environment, and selective activation of the dye in the proper environment. Targeting of pH sensors can be used to assess protein localization to acidic compartments such as endosomes[22], and targeting of lipid probes can be used to detect membrane associated proteins fluorogenically, as recently demonstrated with SNAP-tag directed Nile Red[23]. …”

Section: Introductionmentioning

confidence: 99%

Dark dyes–bright complexes: fluorogenic protein labeling

Bruchez

2015

Current Opinion in Chemical Biology

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Complexes formed between organic dyes and genetically encoded proteins combine the advantages of stable and tunable fluorescent molecules and targetable, biologically integrated labels. To overcome the challenges imposed by labeling with bright fluorescent dyes, a number of approaches now exploit chemical or environmental changes to control the properties of a bound dye, converting dyes from a weakly fluorescent state to a bright, easily detectable complex. Optimized, such approaches avoid the need for removal of unbound dyes, facilitate rapid and simple assays in cultured cells and enable hybrid labeling to function more robustly in living model organisms.

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Direct pH Measurements by using Subcellular Targeting of 5(and 6-) Carboxyseminaphthorhodafluor in Mammalian Cells

Cited by 29 publications

References 26 publications

Chemical tags: Applications in live cell fluorescence imaging

Chemical tags: Applications in live cell fluorescence imaging

How to obtain labeled proteins and what to do with them

Dark dyes–bright complexes: fluorogenic protein labeling

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