共查询到20条相似文献,搜索用时 8 毫秒
1.
Cyanine dye labeling reagents containing isothiocyanate groups 总被引:8,自引:0,他引:8
New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry. 相似文献
2.
Cyanine dye labeling reagents for sulfhydryl groups 总被引:8,自引:0,他引:8
Cyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from less than 500 nm to greater than 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity. 相似文献
3.
Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling.The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDASE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling. 相似文献
4.
H Yu J Chao D Patek R Mujumdar S Mujumdar A S Waggoner 《Nucleic acids research》1994,22(15):3226-3232
Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps. 相似文献
5.
In this contribution, we describe the effects of amide coupling reactions on the physical properties of thermoresponsive hydrogel microparticles (microgels). These microgels, when treated via aqueous carbodiimide/sulfo-succinimide coupling protocols, displayed a dramatic modulation of the microgel phase transition thermodynamics. UV spectrophotometry was used to determine that this modulation was due to remarkably stable hydrogel conjugates of sulfo-NHS that resisted degradation under standard hydrolysis protocols. These intermediates result in a shift of the phase transition, along with a large increase in equilibrium microgel swelling degree, due to an increase in chain-chain Coulombic repulsion. Only aggressive hydrolysis protocols resulted in the recovery of the native microgel phase transition, suggesting that an unusually stable succinimidyl ester is formed in the microgel during coupling. 相似文献
6.
Carreon JR Stewart KM Mahon KP Shin S Kelley SO 《Bioorganic & medicinal chemistry letters》2007,17(18):5182-5185
A series of fluorescent compounds suitable for live cell imaging is described. Functionalized forms of four different asymmetric cyanine dyes are reported that are amenable to peptide conjugation. The photophysical properties of the modified dyes and conjugates and the use of the compounds as cellular imaging agents are described. The results obtained indicate that these spectrally versatile compounds, which have absorption and emission profiles spanning the visible spectrum, are useful probes for cellular imaging. 相似文献
7.
J. S. Huebner 《The Journal of membrane biology》1978,39(2-3):97-132
Summary Flash illumination alters the voltage across bilayer lipid membranes in the presence of certain cyanine dyes. The waveforms of the photo-voltage vary systematically with dye structure and imposed transmembrane voltage. Experimental results are reported for 27 positively charged cyanine dyes, primarily oxazole derivatives, using lecithin/oxidized cholesterol bilayer membranes and 10-mm sodium chloride solutions. Several dyes do not induce any photo-voltages. Examples are 3,3 diethyl 9 ethyl 2,2 oxacarbocyanine iodide, 3,3 diethyl 2 oxa 2 thiacyanine iodide, and 3,3 dimethyl 2,2 indocarbocyanine iodide. Several dyes, when added to one side of the membranes, induce monophasic waveforms. Examples are 3,3 dimethyl 2,2 oxacarbocyanine chloride, and 3,4,3,4 tetramethyl 2,2 oxazalinocarbocyanine iodide. Other dyes induce a photo-voltage only if transmembrane voltages are imposed. These waveforms are biphasic with some dyes (3,3 diethyl 2,2 oxacarbocyanine iodide, for example) and monophasic with other dyes (3,3 dibutyl 2,2 oxacarbocyanine iodide, for example).The photo-voltage waveforms are explained by models that consider the movement of charged dye molecules within the membrane, following optical excitation. The dye movements are probably induced through charge rearrangements in the dye associated with long-lived triplet states, isomerization, or through excimer formation. These results provide information on the location and orientation of the dye molecules within bilayer membranes. The variations which occur in the waveforms with applied voltage indicate that these membranes are fluid in the direction perpendicular to the membrane plane. 相似文献
8.
Junyan Han Myung Shin Han Ching-Hsuan Tung 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases.Methods
A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye.Results
AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7.Conclusions
A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity.General Significance
The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging. 相似文献9.
《Journal of biochemical and biophysical methods》2008,70(5):727-733
We ascertained the ability to detect fibrillar β-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins. 相似文献
10.
Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester 总被引:2,自引:0,他引:2
This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry. 相似文献
11.
The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential. 相似文献
12.
D E Wolf 《Biochemistry》1985,24(3):582-586
We have found that 2,4,6-trinitrobenzenesulfonate (TNBS) is an effective quencher of the fluorescence of the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanines (CNdiI's). This quenching appears to occur by complex formation rather than a collisional mechanism. By use of this quenching, we have studied the transbilayer asymmetry of CNdiI labeling for large unilamellar membranes of egg phosphatidylcholine and the plasma membranes of human red blood cells and of ram epididymal spermatozoa. When CNdiI is added to membranes by ethanolic injection, only the outer leaflet labels. We have further shown that in large unilamellar vesicles of egg phosphatidylcholine, the CNdiI's do not appreciably "flip-flop" to the inner leaflet for at least 4 h at temperatures between 4 and 37 degrees C. 相似文献
13.
14.
A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250 nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2 pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments. 相似文献
15.
A versatile fluorescence labeling method employing support bound nitrophenyl, N-hydroxysuccinimidyl, 1-hydroxybenzotriazoyl and oxime active esters is described. The relative kinetic properties of different esters were systematically compared using an environment-independent benzoquinoline dye. 相似文献
16.
To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using F?rster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. 相似文献
17.
We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis. 相似文献
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19.
Barbarella G Zambianchi M Ventola A Fabiano E Della Sala F Gigli G Anni M Bolognesi A Polito L Naldi M Capobianco M 《Bioconjugate chemistry》2006,17(1):58-67
The synthesis of multicolor fluorescent oligothiophene N-succinimidyl esters (TSEs) is reported, and their optical properties are discussed with the aid of ab initio calculations. The esters were coupled to proteins and to 3'-amino-modified oligonucleotides in mild conditions and with similar modalities. A comparative study of the bioconjugate of IgG1 anti-CD3 antibody labeled with a blue fluorescent TSE and with fluorescein isothiocyanate (FITC) is reported, showing that the former achieves higher photoluminescence intensity and optical stability than the latter. Fluorescence resonance energy transfer experiments with TSE-labeled oligonucleotides and examples of cellular imaging via TSE-labeled proteins are reported. 相似文献
20.
The sulfonation reagent, a succinimidyl ester of 3-sulfobenzoic acid, has been synthesized for effective peptide sequencing. It is capable of incorporating an additional mobile proton into the peptide backbone, thus, facilitating efficient collision-induced dissociation. This reagent is easily and inexpensively prepared in short time. Tandem mass spectra of the guanidinated and reagent-sulfonated peptides consist mainly of the y-ion series with higher intensities than those observed for solely guanidinated peptides. These enhanced tandem MS attributes significantly improved MASCOT total-ion scores, thus, allowing more confident peptide sequencing. This derivatization was also very effective for the analysis of tryptic digest of human blood serum proteins separated by two-dimensional gel electrophoresis. When used in LC-MALDI/MS/MS format, this type of derivatization does not adversely affect chromatographic efficiencies. 相似文献