Except for alexa fluor 633, which is a sulfonated rhodamine derivative, all of these new alexa fluor dyes are sulfonated carbocyanine molecules. High quality alexa fluor conjugated secondary antibodies abcam. Alexa fluor dyes are frequently used as cell and tissue labels in fluorescence microscopy and cell biology. An example of a fluorochrome with high quantum yield is alexa fluor 488 which has. The alexa fluor 660 dye exhibits good spectral separation from orange fluorophores, including the alexa fluor 555 dye and phycoerythrin pe, making it a great choice for multicolor applications.
When a fluorophore absorbs a photon of light, an energetically excited state is formed. Preparation note protect from light legal information. Quantitative comparison of longwavelength alexa fluor. Table of fluorochromes salk institute for biological studies. Fluorescence quantum yield is a measurement of emission efficiency and is calculated by the formula. Like other alexa fluor dyes, alexa fluor 633 exhibits uncommon photostability, making it an ideal choice for fluorescent microscopy. This is a table of some characteristics of fluorochromes useful for flow cytometry or fluorescence microscopy. Alexa fluor 633 dye molecules can be attached to proteins at high molar ratios without significant selfquenching, enabling brighter conjugates and more sensitive detection. For alexa fluor 488, alexa fluor 532, alexa fluor 546, alexa fluor 555, alexa fluor 568, alexa fluor 594 and alexa fluor 647 dyes, qy measurements were made in pbs 50 mm potassium phosphate, 150 mm nacl, ph 7. Alexa 350, alexa 430, alexa 488, alexa 532, alexa 546, alexa 568, and alexa 594 dyes are a new series of fluorescent dyes with emissionexcitation 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.
Alexa fluor 633, alexa fluor 647, cy5, dylight 633 yields the brightest antibody conjugates among spectrally similar dyes using 633 nm hene laser or the 635 nm red diode laser far more photostable than alexa fluor 647 highly watersoluble 15 cf640r 642 662 633, 635, or 640 nm laser alexa fluor 647, atto 647n, cy5. Properties and structures a range of alexa dyes is available, with emission wavelengths spanning the visible spectrum and extending into. Through fret, the dye is able to borrow brightness from the qd, and the fret efficiency can be tuned through the assembly of multiple acceptor dyes per qd. A large number within the range is preadsorbed ensuring low species crossreactivity thus ideal for multicolor analysis. They are conjugated postsynthesis to an aminomodified oligo as nhs esters. Cf633 630 650 633 or 635 nm laser alexa fluor 633, alexa fluor 647, cy5, dylight 633 yields the brightest antibody conjugates among spectrally similar dyes using 633 nm hene laser or the 635 nm red diode laser far more photostable than alexa fluor 647 highly watersoluble 15 cf640r 642 662 633, 635, or 640 nm laser. Yields the brightest anibody conjugates among spectrally similar dyes when excited by the 633 nm hene laser or the 635 nm red diode laser.
Alexa fluor568 578 603 532 red 6 480,565 6 texas red 595 6 568,543,514 hilyte fluortm 594 593 616 petexas red 566 616 alexa fluor594 590 617 dylight594 593 618 evitag tm quantum dotsmaplered orange 600 620 alexa fluor610 612 628 chromeo tm 494 494 628 alexa fluor633 632 647 surelightapc 652 657 dylight633 638 658. Substance name solvent ph excitation max nm emission max nm qdot 705. Fluoroquest fluorescence quantum yield determination kit optimized for. The fluorescence quantum yield of alexa fluor 532 carboxylic acid, succinimidyl ester in 50 mm potassium phosphate, 150 mm nacl ph 7. Western blot analysis of rat kidney tissue lysates showing detection of nkcc2 protein using rabbit antinkcc2 polyclonal antibody spc401. Alexa fluor 633 conjugates can be used in multicolor flow cytometry with instruments equipped with a second red laser or red diode. Within groups, roughly in order of excitation wavelength families excepted. Alexa fluor 633 621 639 1200 molecular probes alexa fluor 647 650 668 1250.
Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. Fqy fluorescence quantum yield in aqueous buffer ph 7. At working concentrations, the quantum yield and colloidal dispersion of these materials has been found to be remarkably stable across ph 69 not investigated outside this range in tris, hepes, phosphate, and borate buffers. Fluoroquest fluorescence quantum yield determination kit optimized for bioconjugates catalog number. Name max absorption max emission extinction coefficient quantum yield brightness index conditions. F 1 signifies a 100% efficient fluorescence process. A lower yield may result from polymodifications andor strong secondary structures minimum guaranteed yields. Alexa 350, alexa 430, alexa 488, alexa 532, alexa 546, alexa 568, and alexa 594 dyes are a new series of fluorescent dyes with emissionexcitation 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.
Alexa fluor dyes antibody conjugates molecular probes. The quantum yield is a readout of the efficiency of the fluorescence process. The absorption maximum of alexa fluor 546 occurs near 556 nm, and the emission maximum is near 573 nm. Human vision is insensitive to light beyond 650 nm, and therefore it is not possible to view the farredfluorescent dyes by looking. Aminereactive nhydroxysuccinimidyl 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. Fluorescence emission of the alexa fluor 680 dye is well separated from. These postsynthesis manually coupled dyes are available. Handbook of fluorescent dyes and probes wiley online books. Quantitative comparison of longwavelength alexa fluor dyes. Antinkcc2 antibody spc401 rabbit polyclonal stressmarq. Atto633 can be used as a substitute dye for dy633, lightcycler red 640, alexa fluor 633, and cy 5. They are available in a wide spectral range from blue to deep red. Probes with high fluorescence quantum yield and high photostability allow detection. The next generation fluorescent dyes for labeling proteins and.
Alexa fluor dyes spanning the visible and infrared spectrum. These postsynthesis manually coupled dyes are available in a wide spectral range from blue to deep red. Fluorochromes description cincinnati childrens hospital. The dye can be efficiently excited by the 633 or 635 nm laser with emission maximum at 650 nm. Alexa fluor 647 dye is a bright, farredfluorescent dye with excitation ideally suited for the 594 nm or 633 nm laser lines. A guide to recording fluorescence quantum yields introduction. Atto633 has a strong absorption at 629nm, high fluorescence at 657nm extinction coefficient 1. The commonly used and very bright alexa fluor 488 fluorophore. Cf633 14 630 650 633 or 635 nm alexa fluor 633, alexa fluor 647, cy5, dylight 633 yields the brightest antibody conjugates among spectrally similar dyes far more photostable than alexa fluor 647 compatible with superresolution tirf, fiona, and gshrimp cf640r 15 642 662 633, 635, or 640 nm. Hydrazide forms of the alexa dyes are very bright, formaldehydefixable polar tracers. Other properties listed for this fluorescent dye include the physical form, solubility, absorption maxima, emission maxima, molar extinction.
For stable signal generation in imaging and flow cytometry, alexa fluor 647 dye is phinsensitive over a wide molar range. Alexa dyes, a series of new fluorescent dyes that yield. Molecular probes like alexa fluor 700 conjugates are useful in labeling and detecting specific properties of. Alexa fluor dyes sulfonation of coumarin rhodamine cyanine dyes. Visualization of qdot conjugates using a custom filter set is preferred because excitation and detection is less. Properties and structures a range of alexa dyes is available, with emission wavelengths spanning the visible spectrum and extending into the infrared region of the spectrum. Alexa fluor 700 is a molecular probe exhibiting a red color which absorbs light at 702 nm. In principle, the fluorescence intensity from acceptor dyes assembled to. F number of emitted photonsnumber of absorbed photons. Cf 633 maleimide suitable for fluorescence sigmaaldrich. Alexa fluor 700 far red 696 633, 635, 640 719 bd horizon bv711 far red 407 405 711 bd horizon buv737 far red 348 355 737. Material amount concentration storage stability alexa fluor dye succinimidyl ester or tfp ester component a 3 vials na. Cf633 is a novel rhodaminebased farred dye with excellent fluorescence quantum yield and exceptional photostability.
The alexa fluor 633, alexa fluor 647, dy630, and dy635 dyes can. Alexa fluor dyes can be conjugated directly to primary antibodies or to secondary antibodies to amplify. Alexa fluor oligonucleotide amine labeling kits table 1. The fate of this species is varied, depending upon the exact nature of the fluorophore and its surroundings, but the end result is deactivation loss of energy and return to the ground state. Bd biosciencesfluorochrome reference chart w d stain index dw resolution sensitivity the ability to resolve a dim positive signal from background is a function of the difference between positive and background peak means d and the spread of the. Eugene, or with the ses of the cy series of dyes and some other currently available longwavelength dyes.
Atto700 has a strong absorption peak at 690nm, with. The commonly used and very bright alexa fluor 488 fluorophore for example has a high quantum yield of 0. Probes with high fluorescence quantum yield and high photostability allow detection of lowabundance biological structures with great sensitivity. The alexa fluor dyes have been conjugated to a large number of secondary antibodies raised in different species including donkey, goat and rabbit. Quantitative comparison of longwavelength alexa fluor dyes to. Handbook of fluorescent dyes and probes is the most comprehensive volume available on the subject, covering all the available dyes and probes known to date in the literature for uses in various fields. Alexa dyes have high quantum yield and are relatively photostable. Alexa 488 carboxylic acidsuccinimidyl ester, alexa 532 carboxylic acidsuccinimidyl ester, alexa 532 c 5 maleimide, alexa 546 carboxylic acidsuccinimidyl ester, alexa 555 c 2 maleimide, alexa 568 hydrazide, alexa 594 c 5 maleimide, alexa 633 c 5 maleimide, alexa 647 carboxylic acidsuccinimidyl ester, alexa 647 c 2 maleimide, bodipy. If someone bothered to do it, heshe could go over all published alexa flour structures and calculate proper molecular masses, citing the proper references. The supporting information is available free of charge on the acs publications website at doi. Spectra of available fluorescent dyes isogen life science. Invitrogen alexa fluor 647 dye is a bright, farredfluorescent dye with excitation ideally suited for the 594 nm or 633 nm laser lines. Both the excitation and emission wavelengths of cf633 are shorter than those of the cyaninebased dye cy 5 or alexa fluor 647. Alexa fluor 680 carboxylic acid succinimidyl ester.
It is detected in the fl4 detector of the cores upgraded 2laser facscans. However, the loss of fluorescence of the conjugated cy dyes gruber et. The fluorescence quantum yield of alexa fluor 546 carboxylic acid, succinimidyl ester in 50 mm potassium phosphate, 150 mm nacl ph 7. The most reliable method for recording fluorescence quantum yield is the comparative method of williams et al ref 1. A 100% efficient fluorescence process would have a quantum yield of 1 the maximum quantum yield possible. The alexa fluor 660 dye is efficiently excited by the 633 nm and 647 nm spectral lines of the hene laser and the kryptonion laser, respectively. The relative quantum yield rqy, the integrated photon emission relative to that of an appropriate dye standard of each conjugate was calculated using the following standard dyes.
Alexa fluor 700 emits light at 775 nm, and has a molar mass of approximately 0 gmol. Alexa fluor fluoronanogold contains an extremely uniform 1. Fluoroquest fluorescence quantum yield determination kit. These conjugates were compared with spectrally similar protein conjugates of the cy3, cy5, cy5. The alexa fluor family of fluorescent dyes is a series of dyes invented by molecular probes, now a part of thermo fisher scientific, and sold under the invitrogen brand name.
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