HiLyte™ Fluor Dyes - superior fluorescent labeling dyes.
HiLyte Fluor Dyes>

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.

Key Features

  • Not affected by pH (4-11)
  • Can be paired with proprietary QXL Quenchers for FRET
  • Cost effective

Applications

  • 2-photon/confocal microscopy
  • Flow cytometry
  • In-vivo imaging

Ex (nm)

Em (nm)

Ex Coef (e)
(M-1cm-1)

Fluorescence Quantum
Yield (f)

HiLyte™ Fluor 405

404

428

35,000

0.54

HiLyte™ Fluor 488

497

525

70,000

0.91

HiLyte™ Fluor 555

550

566

150,000

0.10

HiLyte™ Fluor 594

593

616

80,000

0.90

HiLyte™ Fluor 647

649

672

250,000

0.33

HiLyte™ Fluor 680

688

700

190,000

0.36

HiLyte™ Fluor 750

750

782

275,000

0.12



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).




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.
  • 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 Dye Amine C2 Maleimide Hydrazide Succinimidyl Ester (SE)

HiLyte™ Fluor 405

89318
(1 mg)

89320
(1 mg)

89319
(1 mg)

89317-1
(1 mg)
89317-5
(5 mg)

Related Products:

AnaTag™ HiLyte™ Fluor 405 Protein Labeling Kit

HiLyte™ Fluor 405 labeled Secondary Antibodies & Streptavidin

HiLyte™ Fluor 488 Dye

...an Excellent Replacement for FITC


  • Fluorescence of HiLyte™ Fluor 488 is not affected by pH (working pH range is 4-10), a major improvement over fluorescein, which emits its maximum fluorescence only at pH above 9.
  • Protein conjugates prepared with HiLyte™ Fluor 488 dyes are far more superior to conjugates of FITC (Figure 5).
  • HiLyte™ Fluor 488 conjugates are significantly brighter than fluorescein conjugates and are much more photostable.
  • Ex/Em = 497/525 nm, extinction coefficient is 70,000 M-1cm-1, quantum yield of 0.91.

Figure 4. 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). Figure 5. Photostability of HiLyte™ Fluor 488 compared to FITC and FAM (A & B).

HiLyte™ Fluor Dye Acid Amine C2 Maleimide Hydrazide Succinimidyl Ester (SE)

HiLyte™ Fluor 488

81160
(10 mg)

81162
(1 mg)

81164
(1 mg)

81163
(1 mg)

81161-1
(1 mg)

81161
(5 mg)

Related Products:

AnaTag™ HiLyte™ Fluor 488 Protein Labeling Kit

HiLyte™ Fluor 488 labeled Secondary Antibodies & Streptavidin


HiLyte™ Fluor 555 Dye


...an Excellent Replacement for Cy3™ and Alexa Fluor™ 555
  • 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.
  • Ex/Em = 550/566 nm, extinction coefficient is 150,000 M -1cm-1, quantum yield of 0.10.
  • Photostability of HiLyte™ Fluor 555 provides additional time for image capture.


Figure 6. Comparison data of HiLyte™ Fluor 555 and Cy3™ emission at different dye to protein ratio . Figure 7. 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).

HiLyte™ Fluor Dye Acid Amine C2 Maleimide Hydrazide Succinimidyl Ester (SE)

HiLyte™ Fluor 555

81250
(5 mg)

81252
(1 mg)

81254
(1 mg)

81253
(1 mg)

81251
(1 mg)

Related Products:

AnaTag™ HiLyte™ Fluor 5555 Protein Labeling Kit

HiLyte™ Fluor 555 labeled Secondary Antibodies & Streptavidin


HiLyte™ Fluor 594 Dye

...an Excellent Replacement for Texas Red®

  • HiLyte™ Fluor 594 has better labeling performance and stability properties than Texas Red®.
  • 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).
  • Ex/Em = 593/616 nm
  • 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 (validated in immunofluorescence staining).
  • 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.



Figure 8. 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).

HiLyte™ Fluor Dye Acid Amine C2 Maleimide Hydrazide Succinimidyl Ester (SE)

HiLyte™ Fluor 594

81271
(10 mg)

81273(1 mg)

81275
(1 mg)

81274
(1 mg)

81272-1
(1 mg)
81272-5
(5 mg)


Related Products:

AnaTag™ HiLyte™ Fluor 594 Protein Labeling Kit

HiLyte™ Fluor 594 labeled Secondary Antibodies & Streptavidin

HiLyte™ Fluor 647 Dye

...an Excellent Replacement for Cy5™
and Alexa Fluor™ 647

  • Spectra of HiLyte™ Fluor 647 conjugates are only slightly red-shifted compared to Cy5™, thus HiLyte™ Fluor 647 dye is 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.
  • 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, unlike Cy5™ dyes.
  • Ex/Em = 649/672 nm, Extinction coefficient is 250,000 M -1cm-1, quantum yield of 0.33.

  • Spectra of HiLyte™ Fluor 647 conjugates are only slightly red-shifted compared to Cy5™, thus HiLyte™ Fluor 647 dye is 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.

  • 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, unlike Cy5™ dyes.

  • Ex/Em = 649/672 nm, Extinction coefficient is 250,000 M -1cm-1, quantum yield of 0.33.

Figure 9. Performance of dye-goat anti-rabbit IgG conjugates (left). HiLyte™ Fluor 647 and Alexa Fluor™ 647 photostability over time (right).


A B
Figure 10. 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).

HiLyte™ Fluor Dye Acid Amine C2 Maleimide Hydrazide Succinimidyl Ester (SE)

HiLyte™ Fluor 647

81255
(5 mg)

81257
(1 mg)

81259
(1 mg)

81258
(1 mg)

81256
(1 mg)

Related Products:

AnaTag™ HiLyte™ Fluor 647 Protein Labeling Kit

HiLyte™ Fluor 647 labeled Secondary Antibodies & Streptavidin


HiLyte™ Fluor 680 Dye

...an Excellent Replacement for Cy5.5™

  • 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.
  • Ex/Em = 688/700 nm, Extinction coefficient is 190,000 M -1cm-1, quantum yield of 0.36.

HiLYte 680

Figure 11. CHO-K1 cells were labeled with rabbit anti-tubulin primary antibody and a secondary antibody goat anti-rabbit IgG conjugated to HiLyte™ Fluor 680 (red); Paxillin EGFP (green) and a nuclear stain, DAPI (blue). Image courtesy of Kimberly Young and Claire M. Brown, McGill University Imaging Facility, Montreal, Canada.

HiLyte™ Fluor Dye Acid Amine C2 Maleimide Hydrazide Succinimidyl Ester (SE)

HiLyte™ Fluor 680

81260
(5 mg)

81262
(1 mg)

81264
(1 mg)

81263
(1 mg)

81261
(1 mg)


HiLyte™ Fluor 750 Dye

...an Excellent Replacement for Cy7™

  • Spectrally similar to Cy7™ dye, HiLyte™ Fluor 750 dye is the longest-wavelength HiLyte™ Fluor dye currently available.
  • Fluorescence emission maximum of HiLyte™ Fluor 750 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.
  • Ex/Em = 750/782 nm, extinction coefficient is 275,000 M -1cm-1, 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.
  • Infrared spectra of HiLyte™ Fluor 750 makes it the ideal candidate for in vitro and in vivo tissue imaging.


HiLYte 680
Figure 12. Western blot 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 (Odyssey, Li-Cor, Inc). Figure 13. CHO-K1 cells labeled with rabbit anti-tubulin primary antibody and a secondary antibody goat anti-rabbit IgG conjugated to HiLyte Fluor™ 750 (red); Paxillin EGFP (green) and a nuclear stain, DAPI (blue). Image courtesy of Kimberly Young and Claire M. Brown, McGill University Imaging Facility, Montreal, Canada.

HiLyte™ Fluor Dye Amine C2 Maleimide Hydrazide Succinimidyl Ester (SE)

HiLyte™ Fluor 750

81267
(1 mg)

81269
(1 mg)

81268
(1 mg)

81266
(1 mg)

Related Products:

AnaTag™ HiLyte™ Fluor 570 Protein Labeling Kit

HiLyte™ Fluor 570 labeled Secondary Antibodies & Streptavidin

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.

 

 

Table 1. AnaTag™ Protein Labeling kits are available in both large scale (3x5mg) and microscale (3x200ug) labeling sizes.

Product

3 x 5 mg

3 x 200 ug

AnaTag™ HiLyte™ Fluor 405 Protein Labeling Kit

72143

72142

AnaTag™ HiLyte™ Fluor 488 Protein Labeling Kit

72047

72048

AnaTag™ HiLyte™ Fluor 555 Protein Labeling Kit

72045

72046

AnaTag™ HiLyte™ Fluor 594 Protein Labeling Kit

72121

72120

AnaTag™ HiLyte™ Fluor 647 Protein Labeling Kit

72049

72050

AnaTag™ HiLyte™ Fluor 680 Protein Labeling Kit

72119

72118

AnaTag™ HiLyte™ Fluor 750 Protein Labeling Kit

72043

72044

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 2. HiLyte™ Fluor labeled secondary antibodies.

Product

HiLyte™ Fluor 405

HiLyte™ Fluor 488

HiLyte™ Fluor 555

HiLyte™ Fluor 594

HiLyte™ Fluor 647

HiLyte™ Fluor 680

HiLyte™ Fluor 750

Goat anti-mouse IgG (H+L)

28175-H405

28175-H488

28175-H555

28175-H594

28175-H647

28175-H680

28175-H750

Goat anti-mouse IgG (H+L), highly cross adsorbed

61057-H405

61057-H488

61057-H555

61057-H594

61057-H647

61057-H680

61057-H750

Goat anti-rabbit IgG (H+L)

28176-H405

28176-H488

28176-H555

28176-H594

28176-H647

28176-H680

28176-H750

Goat anti-rabbit IgG (H+L), highly cross-adsorbed

61056-H405

61056-H488

61056-H555

61056-H594

61056-H647

61056-H680

61056-H750

Rabbit anti-mouse IgG (H+L)

28164-H405

28164-H488

28164-H555

28164-H594

28164-H647

28164-H680

28164-H750

Rabbit anti-goat IgG (H+L)

28168-H405

28168-H488

28168-H555

28168-H594

28168-H647

28168-H680

28168-H750

Rabbit anti-chicken IgY (H+L)

29709-H405

29709-H488

29709-H555

29709-H594

29709-H647

29709-H680

29709-H750

































Table

Table 3. A listing of HiLyte™ Fluor-streptavidin conjugates.

Product

HiLyte™ Fluor 405

HiLyte™ Fluor 488

HiLyte™ Fluor 555

HiLyte™ Fluor 594

HiLyte™ Fluor TR

HiLyte™ Fluor 647

HiLyte™ Fluor 680

HiLyte™ Fluor 750

Streptavidin

Dye Conjugates

60672-H405

60665

60666

60672-H594

60671

60667

60659-H680

60659-H750

Table 4. Examples of some HiLyte™ Fluor labeled primary antibodies.

Product

HiLyte™

Fluor 488

HiLyte™

Fluor 555

HiLyte™

Fluor 594

HiLyte™

Fluor 647

Anti- alpha-Actin (smooth muscle), polyclonal

29553-H488

29553-H555

29553-H594

29553-H647

Anti-GST Tag, monoclonal

29531-H488

29531-H555

29531-H594

29531-H647

Anti-His Tag, polyclonal

29673-H488

29673-H555

29673-H594

29673-H647

Anti-His Tag, monoclonal

61250-H488

61250-H555

61250-H594

61250-H647


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