共查询到20条相似文献,搜索用时 15 毫秒
1.
J E Whitaker R P Haugland P L Moore P C Hewitt M Reese R P Haugland 《Analytical biochemistry》1991,198(1):119-130
Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei. 相似文献
2.
Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. 总被引:15,自引:0,他引:15
N Panchuk-Voloshina R P Haugland J Bishop-Stewart M K Bhalgat P J Millard F Mao W Y Leung R P Haugland 《The journal of histochemistry and cytochemistry》1999,47(9):1179-1188
Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges. 相似文献
3.
Judith E Berlier Anca Rothe Gayle Buller Jolene Bradford Diane R Gray Brian J Filanoski William G Telford Stephen Yue Jixiang Liu Ching-Ying Cheung Wesley Chang James D Hirsch Joseph M Beechem Rosaria P Haugland Richard P Haugland 《The journal of histochemistry and cytochemistry》2003,51(12):1699-1712
Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications. 相似文献
4.
Petchprayoon C Suwanborirux K Tanaka J Yan Y Sakata T Marriott G 《Bioconjugate chemistry》2005,16(6):1382-1389
We present the design, synthesis, and biochemical and spectroscopic characterization of five functional fluorescent conjugates of kabiramide C (KabC), a small molecule biomimetic of gelsolin. The tetramethylrhodamine (TMR), rhodol green (RG), IC5, dapoxyl (DAP), and fluorescein diester (FDE) conjugates of KabC bind specifically to actin at the barbed end in a 1:1 complex. These probes are shown to function in an indistinguishable manner to the unmodified KabC. Various modalities of the fluorescence emission of these KabC probes, including fluorescence anisotropy and fluorescence resonance energy transfer, are used for the development of assays for the rapid determination of G-actin concentration in solution. The TMR-KabC and FDE-KabC probes are cell permeable and provide unique imaging information on the distribution and dynamics of actin filament within living cells. 相似文献
5.
Gibson SL Holt JJ Ye M Donnelly DJ Ohulchanskyy TY You Y Detty MR 《Bioorganic & medicinal chemistry》2005,13(23):6394-6403
Several thio and seleno analogues of tetramethylrosamine (TMR) were prepared. Thio derivatives of TMR have absorption maxima near 570 nm, while seleno derivatives of TMR have absorption maxima near 580 nm. The 3- or 4-N,N-dimethylaminophenyl substituent in the 9-position greatly increases internal conversion, which lowers quantum yields for fluorescence and the generation of singlet oxygen. Thio and seleno analogues of TMR are effective photosensitizers against chemosensitive AUXB1 cells in vitro and against multidrug-resistant CR1R12 cells in vitro, which have been treated with verapamil. The CR1R12 cells accumulated significantly lower concentrations of the photosensitizers relative to the AUXB1 cells presumably due to the expression of P-glycoprotein (Pgp) in the CR1R12 cells. Following treatment with 5 x 10(-5) M verapamil, the uptake in CR1R12 cells of several fluorescent thio analogues of TMR is comparable to that observed for the chemosensitive AUXB1 cells. 相似文献
6.
Boga C Puggioli S Gherpelli M Farruggia G Pagnotta E Masotti L Neyroz P 《Analytical biochemistry》2004,335(2):196-209
Different strategies are presented to conjugate a fluorescein moiety to 9- and 10-hydroxystearic acids (HSAs). 5-Amino-fluorescein (5-AF) was used as a starting reagent. When reacted with acyl-chloride-modified HSAs, 5-AF gave rise to stable amide derivatives with a 75% reaction yield. These products exhibited the typical steady-state and time-resolved fluorescence properties of the fluorescein chromophore with absorption at 494 nm and emission at 519 nm. Flow cytometry studies confirmed the distinct proapoptotic effect of underivatized 9-HSA on Jurkat cells and revealed a comparable ability of its amide derivative. Confocal microscopy imaging studies showed that green fluorescence could stain intracellular membranous structures. Moreover, dual-dye labeling with Mito Tracker Red, followed by colocalization analysis, revealed that HSA can move to the mitochondria. Thus, fluorescent derivatives of HSA can be used to monitor the localization of these biologically active molecules in living cells and can provide a useful tool for linking biochemical investigation with optical visualization methods. In contrast, when unmodified HSAs were used, the reaction gave monoesterified and diesterified fluorescein derivatives. These products exhibited unusual steady-state and time-resolved fluorescence properties with the excitation wavelength at 342 nm and the emission wavelength at 432 nm. It is shown that the synthesized HSA amides of fluorescein provide all of the typical photophysical and instrumental advantages of this popular dye, whereas the unusual luminescence and excitation properties of the monoester and diester of the 5-aminofluorescein would make these dyes interesting to explore as potential candidates for two photon excitation applications. 相似文献
7.
V. E. Kuznetsova V. A. Vasiliskov O. V. Antonova V. M. Mikhailovich A. S. Zasedatelev A. V. Chudinov 《Russian Journal of Bioorganic Chemistry》2008,34(1):130-133
New indodicarbocyanine dyes with the carboxybutyl group in position-3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively. 相似文献
8.
Kuznetsova VE Vasiliskov VA Antonova OV Mikhaílovich VM Zasedatelev AS Chudinov AV 《Bioorganicheskaia khimiia》2008,34(1):141-144
New indodicarbocyanine dyes with the carboxybutyl group in position 3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively. 相似文献
9.
High sensitivity of Stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge 总被引:4,自引:0,他引:4
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Sensitivity spectra of Stark-shift voltage sensitive dyes, such as ANNINE-6, suggest the use of the extreme red edges of the excitation spectrum to achieve large fractional fluorescence changes with membrane voltage. This was tested in cultured HEK293 cells. Cells were illuminated with light at the very red edge of the dye's excitation spectrum, where the absorption cross section is as much as 100 times smaller than at its peak. The small-signal fractional fluorescence changes were -0.17%/mV, -0.28%/mV, and -0.35%/mV for one-photon excitation at 458 nm, 488 nm, and 514 nm, respectively, and -0.29%/mV, -0.43%/mV, and -0.52%/mV for two-photon excitation at 960 nm, 1000 nm, and 1040 nm, respectively. For large voltage swings the fluorescence changes became nonlinear, reaching 50% and -28% for 100 mV hyper- and depolarization, respectively, at 514 nm and 70% and -40% at 1040 nm. Such fractional sensitivities are approximately 5 times larger than what is commonly found with other voltage-sensing dyes and approach the theoretical limit given by the spectral Boltzmann tail. 相似文献
10.
Studies of polypeptide structure by fluorescence techniques. 3. Interaction between dye and macromolecule in fluorescent conjugates 总被引:1,自引:0,他引:1
The effects of pH on the polarization of fluorescence of dyes dissolved in media of high viscosity or conjugated to polypeptides that undergo no structural transitions indicate that DNS is useful for studying pH-dependent molecular transition over the range pH 2.5–14, whereas fluorescein is useful only over the range pH 6–8. Heating and cooling in aqueous solutions cause no change in the polarization of fluorescein or of DNS; therefore, the dyes themselves do not introduce artifacts into heating studies of the dye conjugates. The interaction between fluorescein or DNS and the molecule to which it is conjugated varies and thus may affect the measurements made with the conjugates: the rotational relaxation times of polylysine, of a copolymer of glutamic acid and lysine, and of lysozyme are approximately twice as long when measured with DNS-conjugates as when measured with fluorescein-conjugates. The explanation for this observation is postulated to lie in the tighter binding between fluorescein and the molecule to which it is conjugated, presumably around the point of its covalent attachment, which makes it a better indicator of the behavior of the rotational kinetic unit of the polypeptide chain. The stronger binding of fluorescein is inferred from two lines of evidence: (1) the fluorescent intensity and ultraviolet spectra of a fluorescein–polylysine conjugate are less susceptible to changes in solvent than those of the DNS conjugate, and (2) the net charge of the polypeptide affects the ionization of fluorescein much less than it affects the ionization of DNS. Additional evidence from previous studies corroborates this conclusion. Thus, it is important to establish the relationship between the fluorescent dye and the molecule to which it is conjugated before using the fluorescence data to calculate rotational relaxation times and other molecular parameters. 相似文献
11.
BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially termed the CryptoFluortrade mark dyes) for their applicability to flow cytometry, both in extracellular and intracellular labeling applications. METHODS: Several cell lines were labeled with biotin-conjugated antibodies against expressed extracellular surface proteins, followed by streptavidin conjugates of three cryptomonad phycobiliproteins (CryptoFluor-2, CryptoFluor-4, and CryptoFluor-5). Cells were then analyzed by flow cytometry using a variety of laser lines and emission filters to establish the optimal excitation/emission characteristics for each fluorochrome. Some cells were permeabilized and labeled for intracellular antigens, also using the cryptomonad fluorochromes. Where appropriate, parallel samples were labeled with other fluorochromes (including R-PE, APC, the cyanin dyes Cy3 and Cy5, and others) to gauge the performance of the cryptomonad fluorochromes against fluorescent labels previously evaluated for flow cytometry. RESULTS: CryptoFluor-2 possessed excitation/emission maxima similar to those of APC and Cy5, with good excitation in the red (HeNe laser 632 nm) and strong emission in the far red (660 nm). CryptoFluor-4 possessed excitation/emission maxima similar to those of Cy3, with optimal excitation in the green (Kr 530 nm) and strong emission in the yellow/orange (585 nm). CryptoFluor-5 possessed excitation/emission maxima similar to those of lissamine rhodamine, with optimal excitation in the yellow (Kr 568 nm) and emission in the orange (610 nm). All cryptomonad fluorochromes gave satisfactory results for both intracellular and extracellular labeling, with detection sensitivities that were comparable or better than traditional phycobiliproteins and low- molecular weight synthetic fluorochromes such as the cyanin dyes. CONCLUSIONS: The CryptoFluor fluorochromes were applicable to flow cytometric immunodetection, with excitation and emission conditions commonly found on multilaser instruments. Performance of several of these dyes was at least comparable to existing fluorescent labels. The low molecular weights (30-60 kd) of phycobiliproteins may make them particularly useful in intracellular antigen detection. Cytometry 44:16-23, 2001. Published 2001 Wiley-Liss, Inc. 相似文献
12.
Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle. 相似文献
13.
Several new derivatives of fluorescein were synthesized. The dyes were characterized by NMR; and the absorbance, excitation, and emission spectra were measured. The fluorescence quantum yields of the dyes were determined. The pKa3 values of the dyes were measured by fluorescence titration. The characteristics of the fluorescein and sulfonefluorescein derivatives were compared. The most promising dye for use in cell analysis appeared to be compound 9, which was given the name Vita Blue. The dibutyrate ester of Vita Blue was made and the compound was given the name Vita Blue dibutyrate (VBDB, 14). The Km of VBDB with pig liver esterase was measured and found to be 4 x 10(-5) M. The pKa3 of Vita Blue was 7.56 +/- 0.03; both acidic and basic forms were fluorescent (dual fluorescence). The use of VBDB as an intramolecular esterase substrate and its utility for the discrimination between live and dead cells by flow cytometry is described. 相似文献
14.
E. E. Belobritskaya M. V. Neunylova V. A. Vasiliskov V. D. Rumyantseva A. V. Chudinov A. S. Zasedatelev 《Russian Journal of Bioorganic Chemistry》2007,33(6):617-619
A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480–530 nm and fluorescence maxima at 500–550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled oligonucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction. 相似文献
15.
Belobritskaia EE Neunylova MV Vasiliskov VA Rumiantseva VD Chudinov AV Zasedatelev AS 《Bioorganicheskaia khimiia》2007,33(6):664-666
A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480-530 nm and fluorescence maxima at 500-550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled olgionucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction. 相似文献
16.
橙色荧光蛋白——绿色荧光蛋白GFPxm的改造 总被引:3,自引:0,他引:3
最近报道了从大型多管水母中分离出新的gfp基因。经大肠杆菌表达并纯化出的绿色荧光蛋白 (GFPxm)具有 4 76nm的激发峰和 4 96nm的发射峰 ,但是只能在低温下成熟的缺点限制了它的应用。这里进一步报道GFPxm的 12种突变型。在大肠杆菌中的表达结果表明 ,有 7种突变型在 37℃条件下产生高的荧光强度。在 2 5、32和 37℃条件下表达 6h ,GFPxm16、GFPxm18和GFPxm19的相对荧光强度均高于增强型绿色荧光蛋白 (EGFP) ,而GFPxm16和GFPxm16 3在 4 2℃高温表达时仍能保持高的荧光强度。这 7种突变型中的 4种在哺乳动物细胞中已获得良好表达。此外 ,有 6种突变型的荧光光谱红移 ,目前所达到的最长激发峰为 5 14nm、最长发射峰为 5 2 5nm。另外有 3种突变型具有包括紫外在内的两个激发峰 ,1种突变型只有单一的紫外激发峰。首次报道具有橙色荧光的突变型OFPxm ,它的激发峰为 5 0 9nm、发射峰为 5 2 3nm。 5 2 3nm属于黄绿色 ,但肉眼看到的蛋白为橙色。OFPxm在高温下可得到高水平表达且很好地成熟 ,但是因为低的量子产率而荧光强度相对较低。 相似文献
17.
In this report, the development of a new class of monocarboxylate functionalized cyanine derivatives using improved synthetic procedures is detailed. The employed synthetic strategy relies on efficient nucleophilic attack of alkyl-thiols on cyanine dyes bearing chloro-substituted polymethinic linkers. Monocarboxylate derivatized fluorochromes (CyTE dyes) can be prepared in one step in greater than 90% yield without the need for additional purification. Several of the fluorochromes synthesized by this route show no tendency to aggregate in aqueous solution and have excitation and emission maxima greater than 800 nm. The potential utility of the CyTE fluorochromes was demonstrated through direct labeling of phage displaying a vascular cellular adhesion molecule-1 (VCAM-1) targeting peptide. Endothelial cell internalization of the VCAM-1 targeted phage was monitored via near-infrared fluorescence microscopy. 相似文献
18.
The Sonogashira reaction was used for modifications of borated green fluorescence protein chromophore derivatives, 4-(2-(difluoroboryl)benzylidene)-1H-imidazol-5(4H)-ones, for the development of new fluorescent dyes. The derivatives bearing an acetylene fragment and a difluoroboryl group were obtained in high yields. The modification resulted in a significant bathochromic shift of the absorption and emission maxima and is a promising method for the development of new fluorescent dyes. 相似文献
19.
《Journal of receptor and signal transduction research》2013,33(1-4):333-343
ABSTRACTG protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY® TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY® TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY® TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY® TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 µL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision. 相似文献
20.
Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set. 相似文献