HiLyte™ Fluor Dyes – brilliant


AnaSpec’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.

HiLyte™ Fluor Dye

Ex (nm)

Em (nm)

Extinction Coefficient (e)(M-1cm-1)

Fluorescence Quantum Yield (f)

Fluorescence Life Time (τ) (ns)

HiLyte™ Fluor 405

404

428

35,000

0.54

Not available

HiLyte™ Fluor 488

497

525

70,000

0.91

4.1

HiLyte™ Fluor 555

550

566

150,000

0.10

0.3

HiLyte™ Fluor 594

593

616

80,000

0.90

4.2

HiLyte™ Fluor 647

649

672

250,000

0.33

1.0

HiLyte™ Fluor 680

688

700

190,000

0.36

1.1

HiLyte™ Fluor 750

750

782

275,000

0.12

0.7


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

HiLyte™ Fluor Dye Acid Amine

(1 mg)

C2 Maleimide

(1 mg)

Hydrazide

(1 mg)

Succinimidyl Ester, SE

HiLyte™ Fluor 405

89316

(10 mg)

89318

89320

89319

89317-1
(1 mg)

89317-5
(5 mg)

HiLyte™ Fluor 488

81160

(10 mg)

81162

81164

81163

81161-1
(1 mg)

81161
(5 mg)

HiLyte™ Fluor 555

81250

(5 mg)

81252

81254

81253

81251
(1 mg)

HiLyte™ Fluor 594

81271

(10 mg)

81273

81275

81274

81272-1
(1 mg)

81272-5
(5 mg)

HiLyte™ Fluor 647

81255

(5 mg)

81257

81259

81258

81256
(1 mg)

HiLyte™ Fluor 680

81260

(5 mg)

81262

81264

81263

81261
(1 mg)

HiLyte™ Fluor 750

81265

(5 mg)

81267

81269

81268

81266
(1 mg)


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

Structure

Function

Acid Amine reactive, needs activation to SE using a condensing agent prior to conjugation

Amine

Carboxylic or carbonyl reactive

Maleimide

Sulfhydryl (-SH) reactive

Hydrazide

Carbonyl reactive

Succinimidyl esters (SE) or NHS ester

Amine (-NH2) reactive to form stable amide bond

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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 Dye, an Excellent Replacement for Cy3™

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).

HiLyte™ Fluor 594 Dye, an Excellent Replacement for Texas Red®

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.

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).

HiLyte™ Fluor 647 Dye, an Excellent Replacement for Cy5™

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).


HiLyte™ Fluor 680 Dye, an Excellent Replacement for Cy5.5™

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).

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. 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.

Product

Abs / Em
(nm / nm)

3 x 5 mg

3 x 200 ug

AnaTag™ HiLyte™ Fluor 405 Protein Labeling Kit

407 / 429

72143

72142

AnaTag™ HiLyte™ Fluor 488 Protein Labeling Kit

499 / 523

72047

72048

AnaTag™ HiLyte™ Fluor 555 Protein Labeling Kit

553 / 568

72045

72046

AnaTag™ HiLyte™ Fluor 594 Protein Labeling Kit

596 / 617

72121

72120

AnaTag™ HiLyte™ Fluor TR Protein Labeling Kit

591 / 622

72051

(3x1mg)

72052

AnaTag™ HiLyte™ Fluor 647 Protein Labeling Kit

652 / 669

72049

72050

AnaTag™ HiLyte™ Fluor 680 Protein Labeling Kit

678 / 699

72119

72118

AnaTag™ HiLyte™ Fluor 750 Protein Labeling Kit

754 / 778

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 5. 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


Table 6. 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 7. 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


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