AnaSpec, EGT Group’s proprietary HiLyte Fluor™ dyes are a series of excellent fluorescent labeling dyes with fluorescence emissions that span the full visible and near infrared spectrum. 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. Other features of the HiLyte Fluor™ dyes include:

• 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.
• More photostable than the classic fluorescent dyes such as FITC, Cy3™ and Cy5™.
• 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.
• Highly fluorescent over a broad pH range with little pH sensitivity.
• Well-separated spectra, 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.

Figure 1. Normalized absorbance spectra (left) and normalized fluorescence spectra (right) of HiLyte Fluor™ 405 (1), HiLyte Fluor™ 488 (2), HiLyte Fluor™ 555 (3), HiLyte Fluor™ 594 (4), HiLyte Fluor™ 647 (5), HiLyte Fluor™ 680 (6), and HiLyte Fluor™ 750 (7).

Table 1. Fluorescence properties of the HiLyte Fluor™ dyes.

Table 2. HiLyte Fluor™ dyes are available in different reactive forms.

Table 3. The reactive forms of HiLyte Fluor™ dyes and their functions.

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HiLyte Fluor™ 405 Dye

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.

		Figure 2. Alpha-tubulin in 3T3 cells probed with mouse anti -alpha tubulin antibody, visualized with HiLyte Fluor™ 405 conjugated goat anti-mouse IgG (cat# 28176-H405).
Figure 3. Human PBMC cells were stained with anti-CD3 mAb (0.9 mg/ml) at 1:500 dilution.Secondary antibodies, goat anti-mouse IgG labeled with HiLyte Fluors at 0.5 mg/ml (cat# 61057-H405, 61057-H488, 61057-H647) were used at 1:200 dilution. Filled histogram: secondary antibody staining only control.

HiLyte Fluor™ 488 Dye, an Excellent Replacement for FITC

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.

Figure 4. Photostability of HiLyte Fluor™ 488 compared to FITC and FAM (A & B). Tubulin in human glioblastoma cells was probed with mouse anti-tubulin, visualized with HiLyte Fluor™ 488-conjugated rabbit anti-mouse IgG (Cat# 28164-H488). Nuclei were stained with Hoechst 33342 (Cat# 83218).

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.

Figure 5. Comparison data of HiLyte Fluor™ 555 and Cy3™ emission at different dye to protein ratio (panel A). 3T3 cells incubated with anti-tubulin antibody and goat anti-rabbit antibodies, labeled with HiLyte Fluor™ 555 (B), or Cy3™ (C). Nuclei were stained with Hoechst 33342 (Cat# 83218).

		Figure 6. α-Tubulin in 3T3 cells probed with mouse anti-a-tubulin and visualized with HiLyte Fluor™ 594 conjugated goat anti-mouse IgG (Cat# 28175-H594), nuclei were stained with DAPI (Cat# 83210).

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.

Figure 7. Performance of dye-goat anti-rabbit IgG conjugates (left). HiLyte Fluor™ 647 and Alexa Fluor™ 647 photostability over time (right).

A B

Figure 8. 3T3 cells incubated with anti-tubulin antibody and goat-anti-rabbit antibodies, labeled with HiLyte Fluor™ 647 (A) or Cy5™ (B), nuclei were stained with Hoechst 33342 (Cat# 83218).

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.

Figure 9. α-Tubulin in 3T3 cells probed with mouse anti-tubulin, visualized with HiLyte Fluor™ 680-conjugated rabbit-anti-mouse IgG (Cat# 28164-H680), nuclei stained with DAPI (Cat# 83210).

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.

Figure 10. Western blot analysis of zebrafish lysate (5 dpf) with Z-Fish™ anti-p53 (CT), cat# 55342. An imunoreactive band at ~53 kDa was detected (A); this band was blocked by the immunizing peptide (B). HiLyte Fluor™ 750 –conjugated secondary antibody, cat# 28176-H750 was used and signals detected with an infrared-imaging system (Odyssey, Li-Cor, Inc).

AnaTag™ HiLyte Fluor™ Protein Labeling Kits

The AnaTag™ HiLyte Fluor™ Protein Labeling Kits provide a convenient way to label proteins using the succinimidyl ester (SE) reactive form of the HiLyte Fluor™ dyes. The succinimidyl ester shows good reactivity and selectivity with the aliphatic amines of proteins and forms a carboxamide bond, which is identical to, and is as stable as the natural peptide bond. HiLyte Fluor™-protein conjugates may be used for immunofluorescence staining, fluorescence in situ hybridization, flow cytometry and other biological applications. Each kit has all the essential components for performing the conjugation reaction and for purifying the HiLyte Fluor™-protein conjugates.

Figure 11. The succinimidyl ester (SE) group of the fluorophore reacts with the amino group of lysines on the protein to form a stable carboxamide bond.

Figure 12. Labeling of an amino group (for instance, side chain of lysine) on a biopolymer with a succinimidyl ester of a dye in 4 easy steps using the AnaTag™ Protein Labeling Kits.

Table 4. AnaTag™ Protein Labeling kits are available in both large scale (3x5mg) and microscale (3x200ug) labeling sizes.

HiLyte Fluor™ Labeled Primary, Secondary Antibodies and Streptavidins

AnaSpec's HiLyte Fluor™ dye-IgG conjugates are prepared using optimal fluorophore to protein labeling ratio to ensure high fluorescent signal and uncompromised IgG function. These HiLyte Fluor™ labeled primary (Table 4) and secondary antibodies (Table 5) can be used in applications such as fluorescence microscopy and flow cytometry. AnaSpec also provides labeling service of our catalog or custom antibodies. Labels include these HiLyte Fluor™ dyesor the classic dyes, such as FITC, TAMRA and fluorescent proteins, such as B-PE, R-PE and APC.

Table 5. Examples of some HiLyte Fluor™ labeled primary antibodies.

Table 6. HiLyte Fluor™ labeled secondary antibodies.

Table 7. A listing of HiLyte Fluor™-streptavidin conjugates.

Trademarks: HiLyte Fluor™ (AnaSpec), Alexa Fluor™ (Invitrogen), Cy™ (GE Healthcare), DyLight™ (Pierce).