Technical Information on Probes Conjugated to Affinity-Purified Antibodies and to Other Proteins:

NEW DyLight fluorescent dyes

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DyLight fluorescent dyes are a new family of dyes with improved brightness and photostability. They are better than or comparable to the best fluorescent dyes from other companies. The detection level of any fluorophore-antibody conjugate depends on brightness and photostability of the dye; antibody activity, specificity, and cross-reactivity; and the optimal moles of dye per antibody. A molar saturation curve vs. fluorescence intensity, antibody activity, background level, and/or other parameters has been established for each dye to optimize the level of antibody detection and minimize background. DyLight fluorescent dyes are highly water soluble and remain fluorescent from pH 4 to pH 9.

The following five DyLight fluorescent dyes (Figure 1, Table 1) are currently available from JIR. They cover the most commonly used excitation sources and filter sets from blue to far-red emissions.

Figure 1. Excitation (left) and emission (right) spectra of DyLight fluorescent dyes conjugated to affinity-purified secondary antibodies. The dyes are DyLight 405 (blue), DyLight 488 (green), DyLight 549 (orange), DyLight 594 (red), and DyLight 649 (brown). This figure illustrates the relative shape and position of each fluorophore in the peak region of its excitation and emission following conjugation to antibodies. Quantitative comparisons should not be made since peak heights have been normalized. All spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc.

Fluorophore	Excitation Peak (nm)	Emission Peak (nm)
DyLight 405	400	421
DyLight 488	493	518
DyLight 549	555	568
DyLight 594	591	616
DyLight 649	652	670

Table 1. Peak wavelengths of absorption and emission maxima for DyLight-conjugated, affinity-purified secondary antibodies from Jackson ImmunoResearch. All spectra were obtained with an M-series Spectrofluorometer from Photon Technologies, Inc. Peak wavelengths may vary slightly depending on the spectrofluorometer used in each laboratory.

DyLight 405-conjugated antibodies are excited maximally at about 400 nm and fluoresce with a peak at about 421 nm. They are very bright and photostable, but their use is limited to confocal microscopes equipped with a 405 nm laser and appropriate emission filter (Table 1).

Figure 2. Mouse retinal tissue stained with DyLight 405 (pseudo-colored green)-donkey anti-rabbit IgG (JIR) with rabbit anti-GFAP (DAKO), DyLight 488 (pseudo-colored red)-donkey anti-mouse IgG (JIR) with mouse anti-neurofilament (Abcam), and DyLight 649 (pseudo-colored blue)-donkey anti-goat IgG with goat anti-collagen IV (Chemicon). Sections were mounted in n-propyl gallate-glycerol for imaging with an Olympus Flouview 1000 Laser Scanning confocal microscope equipped with a 405 nm laser and a krypton/argon laser. Images are courtesy of Gabe Luna, Neuroscience Research Institute, UC Santa Barbara.

Under these conditions, it is possible to perform effective 4-color imaging with good color separation, good photostability, and high sensitivity in both aqueous and permanent mounting media. The combination of DyLight 405, DyLight 488, Rhodamine Red-X, and DyLight 649 provides for maximum color separation (Figure 3).

Figure 3. Emission spectra of DyLight 405 (blue), DyLight 488 (green), Rhodamine Red-X (red), and DyLight 649 (brown). This figure illustrates the relative shape and position of each fluorophore in the region of its emission peak following conjugation to antibodies. It shows that effective 4-color imaging can be performed with maximum color separation using these dyes. Quantitative comparisons should not be made since peak heights have been normalized. All spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc.

Other 4-color dye combinations, which may be equally effective but have less color separation, include DyLight 405, DyLight 488, DyLight 549 (or Cy3), and DyLight 649 (Figure 4).

Figure 4. Mouse retinal tissue stained with DyLight 405 (pseudo-colored green)-donkey anti-rabbit IgG (JIR) with rabbit anti-GFAP (DAKO), DyLight 488 (pseudo-colored red)-donkey anti-mouse IgG (JIR) with mouse anti-neurofilament (Abcam), Cy3 (pseudo-colored blue)-donkey anti-chicken IgG (JIR) with chicken anti-vimentin (Chemicon), and DyLight 649 (pseudo-colored white)- donkey anti-goat IgG (JIR) with goat anti-collagen IV (Chemicon). Sections were mounted in n-propyl gallate-glycerol for imaging with an Olympus Flouview 1000 Laser Scanning confocal microscope equipped with a 405 nm laser and a krypton/argon laser. Images are courtesy of Gabe Luna, Neuroscience Research Institute, UC Santa Barbara.

DyLight 405 is not recommended for use in epifluorescence microscopes, nor is it recommended for flow cytometry, because emission filters generally used in flow cytometers are not optimal for DyLight 405.

DyLight 488-antibody conjugates absorb light maximally around 493 nm and fluoresce with a peak around 518 nm (Figure 1 and Table 1). They are brighter than Cy2 and FITC conjugates and similar in brightness to Alexa Fluor 488 conjugates, but may give less background (Figures 2 and 3).

Figure 2. HEp-2 cells were stained with ANA antibody (Bio-Rad) followed by the indicated fluorophores conjugated to comparable secondary antibodies. For fluorescence microscopy cells were mounted in ProLong Gold (Invitrogen) (upper images) or PBS-90% glycerol containing n-propyl gallate (Fluka) as an anti-fading agent (lower images). One-second exposures were taken at 100X with a Nikon D-70 DSLR camera connected to a Nikon LABOPHOT epifluorescence microscope. Fields with the brightest cells for each fluorophore were selected for imaging.

Figure 3. Human peripheral lymphocytes were stained with anti-CD3 and various fluorophore-conjugated secondary antibodies, and were analyzed in a FACSCalibur flow cytometer (Becton Dickinson). DyLight 488 (green)-, FITC (yellow)-, and Cy2 (blue)-conjugated secondary antibodies were from Jackson ImmunoResearch. A comparable secondary antibody conjugated with Alexa Fluor 488 was purchased from Molecular Probes, Inc. The arrow indicates a slight background with the Alexa Fluor 488 conjugate in this experiment.

DyLight 488 conjugates fade less than FITC and Cy2 conjugates in mounting media without anti-fading agents (Figure 4) indicating that the DyLight 488 molecule is inherently more photostable in epifluorescence microscopes. Anti-fading agents added to mounting media do not increase its photostability (Figure 5).

Figure 4. Fluorescence intensity was measured over time and plotted as a percent of the initial intensity for HEp-2 cells labeled with ANA (Bio-Rad) followed by goat anti-human IgG (H+L) conjugated with various fluorophores. The cells were mounted in PBS containing 90% glycerol without anti-fading agents

Figure 5. Fluorescence intensity was measured over time and plotted as a percent of the initial intensity for HEp-2 cells labeled with ANA (Bio-Rad) folowed by goat anti-human IgG (H+L) conjugated with DyLight 488. The cells were mounted in various mounting media.

DyLight 488 conjugated to secondary antibodies is recommended for maximum sensitivity for all immunofluorescence procedures requiring a green-fluorescing dye.

DyLight 549-antibody conjugates absorb light maximally around 555 nm and fluoresce with a peak around 568 nm (Figure 1 and Table 1). They are brighter than TRITC conjugates, and they are about equal in brightness to Cy3 and Alexa Fluor 555 (Figure 6).

Figure 6. HEp-2 cells were stained with ANA antibody (Bio-Rad) and with the indicated fluorophores conjugated to comparable secondary antibodies. For fluorescence microscopy cells were mounted in ProLong Gold (Invitrogen) (upper images) or Vectashield (Vector Labs)(lower images). One-second exposure were taken at 100X with a Nikon D-70 DSLR camera connected to a Nikon LABOPHOT epifluorescence microscope. Fields with the brightest cells for each fluorophore were selected for imaging.

DyLight 549 conjugates are about as photostable as Alexa 555 conjugates and slightly more photostable than Cy3 conjugates when mounted in PBS-90% glycerol without anti-fading agents (Figure 7). Increased photostability may be achieved for all three conjugates by mounting in an anti-fading medium such as ProLong Gold (Figure 8), Vectashield, or PBS-glycerol containing n-propyl gallate.

Figure 7. Fluorescence intensity was measured over time and plotted as a percent of the initial intensity for HEp-2 cells labeled with ANA (Bio-Rad) and goat anti-human IgG (H+L) conjugated with various fluoro-phores. The cells were mounted in PBS containing 90% glycerol without anti-fading agents.

Figure 8. Fluorescence intensity was measured over time and plotted as a percent of the initial intensity for HEp-2 cells labeled with ANA (Bio-Rad) and goat anti-human IgG (H+L) conjugated with various fluoro-phores. The cells were mounted in ProLong Gold (Invitrogen) anti-fading medium. Similar results were obtained when the cells were mounted in Vectashield (Vector Labs) or PBS-90% glycerol containing n-propyl gallate (Fluka).

DyLight 549- and Cy3-conjugated secondary antibodies are recommended for maximum sensitivity in all immunofluorescence detection procedures within the orange-red portion of the visible spectrum.

DyLight 594-antibody conjugates absorb light maximally around 591 nm and fluoresce with a peak around 616 nm (Figure 1 and Table 1). They are noticeably brighter than Alexa 594 conjugates, and much brighter and more water soluble than Texas Red conjugates (Figure 9).

Figure 9. HEp-2 cells were stained with ANA antibody (Bio-Rad) and with the indicated fluorophores conjugated to comparable secondary antibodies. For fluorescence microscopy cells were mounted in ProLong Gold (Invitrogen). Exposures were taken at 100X with a Nikon D-70 DSLR camera connected to a Nikon LABOPHOT epi-fluorescence microscope. Fields with the brightest cells for each fluorophore were selected for imaging, using 1-second exposures for Alexa and DyLight conjugates and a 2-second exposure for Texas Red conjugates. Similar results were obtained with other anti-fading mounting media.

DyLight 594 conjugates are more photostable than Texas Red and about the same as Alexa 594 when mounted in ProLong Gold mounting medium (Figure 10, left). DyLight 594 and Alexa 594 conjugates were less photostable than Texas Red in Vectashield (Figure 10, middle), whereas DyLight 594 conjugates were slightly more photostable than the others in PBS-90% glycerol containing n-propyl gallate (Figure 10, right).

Figure 10. Fluorescence intensity was measured over time and plotted as a percent of the initial intensity for HEp-2 cells labeled with ANA (Bio-Rad) and goat anti-human IgG (H+L) conjugated with various fluorophores. The cells were mounted in ProLong (Invitrogen) (left), Vectashield (Vector Labs)(middle), or PBS-90% glycerol containing n-propyl gallate (Fluka)(right).

DyLight 594-conjugated secondary antibodies are brighter than other red-fluorescing dye conjugates, and they provide more color separation from green fluorescing dyes than DyLight 549, Cy3, or TRITC conjugates. They are the best choice for immunofluorescence detection in the deep red region of the visible spectrum.

DyLight 649-antibody conjugates absorb light maximally around 652 nm and fluoresce maximally around 670 nm (Figure 1 and Table 1). They are brighter than Cy5 (Figure 11) and Alexa 647 conjugates (Figure 12), and they may give less background than Alexa 647 conjugates (Figure 12).

Figure 11. Adjacent sections of human jejunum stained with anti-PGP primary antibody and DyLight 649 (red)-secondary antibody (left) or Cy5 (red)-secondary antibody (right). Notice the more intense red staining with DyLight 649. Courtesy of Dr. Gwen Crabb and Dr. William R. Kennedy, University of Minnesota.

Figure 12. Human peripheral lymphocytes were stained with anti-CD3 and various fluorophore-conjugated secondary antibodies, and were analyzed in a FACSCalibur (Becton Dickinson) flow cytometer. DyLight 649 (green)-, APC (yellow)-, and Cy5 (blue)-conjugated secondary antibodies were from Jackson ImmunoResearch. A similar secondary antibody conjugated with Alexa 647 (red) was purchased from Molecular Probes, Inc. Notice the slight background at arrow in this experiment with the Alexa 647-secondary antibody.

DyLight 649- and APC-conjugated secondary antibodies are the best choice for flow cytometry when secondary antibodies fluorescing at these wavelengths are desired. DyLight 649 conjugates are the best choice for far red-emitting conjugates for multiple-labeling detection with a confocal microscope.

DyLight™ Fluorescent Dyes is a trademark of Thermo Fisher Scientific.

Alexa Fluor® Dyes and ProLong ® Gold Antifade are registered trademarks of Invitrogen Corporation.

VECTASHIELD^® Mounting Medium is a registered trade mark of Vector Laboratories.

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