Now researchers can have the best dyes conjugated to the highest quality and largest number of secondary antibodies!

Advantages

Brighter, More Photostable
Less Background
DyLight fluorescent dyes conjugated to antibodies from Jackson ImmunoResearch (JIR) are brighter and/or more photostable than those which are currently available from JIR, and 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. For each dye Jackson ImmunoResearch establishes a molar saturation curve vs fluorescence intensity, antibody activity, background level, and/or other parameters to optimize the level of antibody detection and minimize background.

Chemical Properties
DyLight fluorescent dyes are highly water soluble and remain fluorescent from pH 4 to pH 9.

Competitive Price
Jackson ImmunoResearch specializes in secondary antibody production and offers more competitive prices for DyLight conjugates than other vendors offering either DyLight or Alexa ® Fluor dye conjugates.

Unparalleled Antibody Quality and Diversity
Jackson ImmunoResearch, the world leader in production of the highest quality secondary antibodies, offers over 250 secondary antibodies, and in addition offers purified immunoglobulins, streptavidin, anti-digoxin, anti-FITC, and anti-biotin. Many secondary antibodies are extensively adsorbed to ensure minimal cross-reactivity to other species for multiple labeling protocols and for minimal background due to cross-reactivity with host Ig on tissues and cells. Jackson ImmunoResearch offers the following four DyLight fluorescent dyes (Figure 1, Table 1), which cover the most commonly used excitation sources and filter sets from green to far-red emissions. DyLight fluorescent dyes emitting in the blue and infra red are currently undergoing testing.

Figure 1. Excitation (left) and emission (right) spectra of DyLight fluorescent dyes conjugated to affinity-purified secondary antibodies. 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 a M-Series spectrofluorometer system from Photon Technology International, Inc.

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

Fluorophore

Absorption Peak (nm)

Emission Peak (nm)

DyLight 488

493

518

DyLight 549

555

568

DyLight 594

591

616

DyLight 649

652

670

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) and with 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). Images 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 after 1 second exposure to light.

Figure 3. Human peripheral lymphocytes were stained with anti-CD3 and various fluorophore-conjugated secondary antibodies, and were analyzed in a FACS Caliber 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 (Figure5).

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) and 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) and goat anti-human IgG (H+L) conjugated with DyLight 488. The cells were mounted in various mounting media.

DyLight 488 conjugated to secondary antibodies from Jackson ImmunoResearch are the best choice 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). Images 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 after 1 second exposure to light.

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-glycerol containing n-propyl gallate (Fluka).

DyLight 549- and Cy3-conjugated secondary antibodies from Jackson Immuno- Research are recommended for maximum sensitivity for all immunofluorescence detection procedures in 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). Images 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 after 1 second exposure to light for Alexa and DyLight conjugates and 2 seconds 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-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-glycerol containing n-propyl gallate (Fluka)(right).

DyLight 594-conjugated secondary antibodies from Jackson ImmunoResearch 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 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 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 FACS-Calibur (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 from Jackson Immuno-Research 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