The hydrophilic property of most of the HiLyte Fluor™ dyes makes conjugations to protein easier, minimizing the use of organic solvents. The resulting conjugates are relatively resistant to aggregation and precipitation during storage. HiLyte Fluor™ dyes also have better labeling performance than the classic fluorescent labeling dyes such as FITC and rhodamines.
AnaSpec HiLyte Fluor™ dyes is available in a variety of reactive forms. HiLyte Fluor™ conjugates exhibit more intense fluorescence than other spectrally similar conjugates of classic fluorescent dyes such as FITC, TAMRA and ROX under similar excitations. They are more photostable than the classic fluorescent dyes such as FITC, Cy3™ and Cy5™. Also, absorption spectra of HiLyte Fluor™ dyes match the principal output wavelengths of common excitation sources such as 488 nm, 555 nm, 633 nm and 647 nm. They are highly fluorescent over a broad pH range with little pH sensitivity.
Well-separated spectra, HiLyte Fluor™ dyes are enabling many options for multicolor detection of multiple parameters simultaneously in flow cytometry analysis. HiLyte Fluor™ dye-antibody conjugates are validated for Western blot, Immunofluorescence staining and flow cytometry. HiLyte Fluor™ dyes and AnaSpec’s proprietary quenchers QXL™ have been used as fluorophore and quencher pairs for fluorescence “resonance energy transfer (FRET). Can be used to make tandem conjugates with fluorescent proteins, such as PE, APC.
HiLyte Fluor™ 405, available in a variety of reactive forms, such as amine-reactive, thiol-reactive and amine-containing derivatives, allows for easy conjugation to biopolymers. HiLyte Fluor™ 405 has spectral characteristics similar to those of Alexa Fluor™ 405 (Invitrogen) and DyLight Fluor™ 405 (Pierce) dyes with excitation and emission wavelength at 404/428 nm in PBS buffer (pH 7.4). Conjugates of HiLyte Fluor™ 405 are well excited by the 407 nm spectral line of the Krypton laser and the 408 nm line of the violet laser diode. HiLyte Fluor™ 405 conjugates, whether conjugated to primary tag, secondary antibodies or streptavidin, are prepared in an optimized fluorophore/protein labeling ratio to ensure high fluorescent signal and uncompromised biological function. Fluorescence signal can be observed at Ex/Em = 404/428 nm.
FITC, the most popular fluorescent labeling dye for preparing green fluorescent bioconjugates, has its limitations, such as severe photobleaching in microscopy imaging and pH sensitivity. However, protein conjugates prepared with HiLyte Fluor™ 488 dyes (Ex/Em=497 nm/525 nm) are far more superior to conjugates of FITC (Figure 2). HiLyte Fluor™ 488 conjugates are significantly brighter than fluorescein conjugates and are much more photostable. Extinction coefficient is 70,000 M-1cm-1, with a quantum yield of 0.91. Additionally, the fluorescence of HiLyte Fluor™ 488 is not affected by pH (working pH range is 4-10). This pH insensitivity is a major improvement over fluorescein, which emits its maximum fluorescence only at pH above 9.
HiLyte Fluor™ 555 conjugates are more photostable and brighter than conjugates of Cy3™, the preferred dye for preparing orange fluorescent bioconjugates. Compared to the spectra of Cy3™ dyes, the spectra of HiLyte Fluor™ 555 conjugates are slightly red-shifted, resulting in an optimal match of filters designed for Cy3™ dyes. Extinction coefficient is 150,000 M-1cm-1, with a quantum yield of 0.10. The photostability of HiLyte Fluor™ 555 provides researchers with additional time for image capture.
HiLyte Fluor™ 594 has spectral characteristics similar to those of Texas Red®, with excitation and emission wavelength at 593/615 nm in PBS buffer (pH 7.4), and 596/617 nm when conjugated to macromolecules. The labeling performance and stability properties of HiLyte Fluor™ 594 are better than those of Texas Red®. The higher extinction coefficient (80,000 M-1cm-1) and a lower correction factor (0.17) of HiLyte Fluor™ 594 are better than those of Alexa Fluor™ 594 (Invitrogen). The fluorescence quantum yield of HiLyte Fluor™ 594 is 0.9 in aqueous solution. HiLyte Fluor™ 594 conjugated streptavidin provides high fluorescence intensity and low background as validated in immunofluorescence staining of mammalian cells. Biomolecules conjugated to HiLyte Fluor™ 594 exhibit little spectral overlap with green-fluorescent conjugates, and can be efficiently excited by 568 nm line of Ar-Kr laser and by the 594 nm line of orange He-Ne laser.
The spectra of HiLyte Fluor™ 647 conjugates are only slightly red-shifted compared to Cy5™, the preferred dye for preparing near-infrared fluorescent bioconjugates. This slight change in absorption spectrum makes HiLyte Fluor™ 647 dye an optimal match of filters designed for Cy5™ dyes. In a side-by-side comparison of antibody conjugates of HiLyte Fluor™ 647 dyes and Cy5™ conjugates (supplied by other companies), the total fluorescence of HiLyte Fluor™ 647 labeled secondary antibodies is significantly higher than that of Cy5™ conjugates. Extinction coefficient is 250,000 M-1cm-1, with a quantum yield of 0.33. Unlike Cy5™ dyes, HiLyte Fluor™ 647 dyes have very little change in absorption or fluorescence spectra when conjugated to most proteins, oligonucleotides and nucleic acids, thus yielding greater total fluorescence at the same degree of substitution.
With a peak excitation at 678 nm and emission maximum at 699 nm, HiLyte Fluor™ 680 dyes are spectrally similar to Cy5.5™ dyes. Fluorescence emission of HiLyte Fluor™ 680 dyes is well separated from that of other commonly used red fluorophores, such as TAMRA, R-phycoerythrin and HiLyte Fluor™ 647 dyes, making it ideal for three and four-color labeling. Extinction coefficient is 190,000 M-1cm-1, with a quantum yield of 0.36.
Spectrally similar to Cy7™ dye, HiLyte Fluor™ 750 dye is the longest-wavelength HiLyte Fluor™ dye currently available. Its fluorescence emission maximum at 778 nm is well separated from commonly used far-red fluorophores, including HiLyte Fluor™ 647, HiLyte Fluor™ 680 or allophycocyanin (APC), facilitating multicolor analysis. Extinction coefficient is 275,000 M<1cm1, with a quantum yield of 0.7. With a peak excitation at ~753 nm, conjugates of HiLyte Fluor™ 750 dyes can be excited by a xenon-arc lamp or dye-pumped lasers operating in the 720–750 nm range. Its near infrared spectra render the HiLyte Fluor™ 750 dye the ideal candidate for in vitro and in vivo tissue imaging.
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