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CF™633 Dye

Far-red fluorescent dyes offer the advantage of ultra sensitive detection because background signal due to autofluorescence in most biological samples is minimal in this spectral region. For many years, the cyanine dye Cy®5 has been the dye of choice for such detection. More recently, Alexa Fluor® 647 has been developed as a better alternative by having brighter fluorescence and higher photostability. Despite the improvement, Alexa Fluor® 647 still lacks sufficient photostability required for many demanding applications. On the other hand, while Alexa Fluor® 633 is photostable, its fluorescence on proteins is very weak.

In fact, it has been a challenging task for dye chemists to develop a far-red fluorescent dye that is both highly fluorescent and photostable for protein and nucleic acid labeling. Using new chemistry, scientists at Biotium have successfully developed CF™633 to overcome these challenges. With its absorption peak at 630 nm, CF™633 is optimally excited by the 633 nm He-Ne laser or the 635 nm red diode laser. Its emission maximum is at 650 nm, which is 15 nm shorter than that of Alexa Fluor® 647 or Cy®5. Although the detection window on most flow cytometers is centered around the emission peak wavelength of Alexa Fluor® 647 or other Cy®5-like cyanine dyes, CF™633 is still significantly brighter (Figure 2).

The most important advantage of CF™633, however, is its unmatched photostability (Figure 3). The combination of superior brightness and photostability make CF™633 the best choice for any detection system using a 633 or 635 nm laser excitation source.

A full selection of reactive dyes, secondary antibodies, antibody labeling kits, and other bioconjugates including phalloidins, Annexin V and α-bungarotoxin are available for CF™ dyes.

View products associated with this technology…

CF™633 conjugates, labeling kits, and reactive dyes

Technical Summary

  • Abs/Em Maxima: 630/650 nm
  • Extinction coefficient: 100,000
  • Molecular weight: ~820
  • Flow cytometry laser line: 633 or 635 nm
  • Microscopy laser line: 633 or 635 nm
  • Direct replacement for: Alexa Fluor® 633, Alexa Fluor® 647, Cy®5 and DyLight™ 649

Advantages

  • Yields the brightest antibody conjugates among spectrally similar dyes when excited by the 633 nm He-Ne laser or the 635 nm red diode laser
  • Far more photostable than Alexa Fluor® 647
  • Highly water-soluble
Figure 1. Absorption and emission spectra of CF™633 conjugated to goat anti-mouse IgG in PBS.
Figure 1. Absorption and emission spectra of CF™633 conjugated to goat anti-mouse IgG in PBS.

Figure 2. Jurkat cells were stained with intracellular CD3 or isotype control followed by goat anti-mouse IgG conjugates. Fluorescence was analyzed on a BD FACS Calibur in the FL4 channel. The bars represent the relative fluorescence of the geometric means of the population of cells.
Figure 2. Jurkat cells were stained with intracellular CD3 or isotype control followed by goat anti-mouse IgG conjugates. Fluorescence was analyzed on a BD FACS Calibur in the FL4 channel. The bars represent the relative fluorescence of the geometric means of the population of cells.
Figure 3. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF™633 (Biotium) or Alexa Fluor® 647 (Invitrogen) goat anti-rabbit IgG conjugates. Cells were imaged using an Olympus mercury arc lamp microscope equipped with a Cy5 filter set and CCD camera. Images were taken at t=0, 1 min and 5 min.
Figure 3. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF™633 (Biotium) or Alexa Fluor® 647 (Invitrogen) goat anti-rabbit IgG conjugates. Cells were imaged using an Olympus mercury arc lamp microscope equipped with a Cy5 filter set and CCD camera. Images were taken at t=0, 1 min and 5 min.