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CF™680 and CF™680R Dyes

CF™680 and CF™680R are two outstanding near-IR dyes excitable at about 680 nm with emission at about 700 nm. The two dyes each have unique properties suitable for different application needs. CF™680 is a highly water-soluble cyanine-based dye with a molecular weight of ~3000.

This dye is excellent for labeling antibodies, producing the brightest fluorescence and highest signal-to-noise ratio in immunostaining among spectrally similar dyes, such as Cy®5.5, Alexa Fluor™ 680, DyLight™ 680 and IRDye® 680. However, because of its relatively large molecular size, CF™680 is not recommended for labeling nucleic acids or relatively small biomolecules, for which our CF™680R is better suited.

CF™680R is a novel rhodamine-based dye with a relatively small molecular weight of 912. The dye is highly fluorescent and, more importantly, extremely photostable. Rhodamine dyes are traditionally known to be bright and photostable. However, it has been a synthetic challenge to make functional rhodamine dyes with long wavelengths and high water solubility necessary for bio-labeling.

Scientists at Biotium have overcome the challenge and invented a new way to make highly water soluble, bright and photostable rhodamine dyes with wavelengths ranging from red to near-IR. These dyes are excellent for labeling proteins, nucleic acids and small bio-molecules. They are ideal for confocal microscopy, single molecule imaging and other applications that demand both brightness and photostability.

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™680 and CF™680R conjugates, labeling kits, and reactive dyes

 

Technical Summary

CF™680

  • Abs/Em Maxima: 681/698 nm
  • Extinction coefficient: 210,000
  • Molecular weight: ~3241
  • Direct replacement for: Alexa Fluor® 680, Cy®5.5

CF™680R

  • Abs/Em Maxima: 680/701 nm
  • Extinction coefficient: 140,000
  • Molecular weight: ~912
  • Direct replacement for: Alexa Fluor® 680, Cy®5.5

Advantages

CF™680

  • The brightest among spectrally similar 680 nm dyes
  • Superior signal-to-noise ratio in immunostaining
  • Highly water-soluble and pH-insensitive

CF™680R

    • The most photostable 680 nm dye
    • suitable for labeling nucleic acids and small bio-molecules
    • Highly water-soluble and pH-insensitive
Figure 1. Absorption and emission spectra of CF™680 and CF™680R conjugated to goat anti-mouse IgG, respectively, in PBS.
Figure 1. Absorption and emission spectra of CF™680 and CF™680R conjugated to goat anti-mouse IgG, respectively, in PBS.
Figure 2. Jurkat cells were stained with isotype or mouse anti-human intracellular CD3 antibody followed by 1 mg of goat anti-mouse IgG conjugated with Cy®5.5, Alexa Fluor® 680, CF™680 or DyLight™680. Fluorescence was detected by a BD FACS Calibur in the FL4 channel. The bars represent the signal-to-noise ratio of CD3 positive fluorescence to isotype using similar degrees of labeling (DOL).
Figure 2. Jurkat cells were stained with isotype or mouse anti-human intracellular CD3 antibody followed by 1 mg of goat anti-mouse IgG conjugated with Cy®5.5, Alexa Fluor® 680, CF™680 or DyLight™680. Fluorescence was detected by a BD FACS Calibur in the FL4 channel. The bars represent the signal-to-noise ratio of CD3 positive fluorescence to isotype using similar degrees of labeling (DOL).
Figure 3. Photostability comparison by microscopy. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF™680R (Biotium), Alexa Fluor® 680 (Invitrogen) or Cy®5.5 (GE Healthcare) 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. Photostability comparison by microscopy. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF™680R (Biotium), Alexa Fluor® 680 (Invitrogen) or Cy®5.5 (GE Healthcare) goat anti-rabbit IgG conjugates. Cells were imaged
using an Olympus mercury arc lamp microscope equipped with a Cy®5 filter set and CCD camera. Images were taken at t=0, 1 min and 5 min.
Figure 4. Photostability comparison by microscopy. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF™680R, CF™F680, Cy®5.5 or Alexa Fluor® 680 (AF680) goat anti-mouse IgG conjugates. Cells were imaged using an Olympus mercury arc lamp microscope equipped with a Cy®5 filter set and CCD camera. The graph illustrates the relative fluorescence intensities of sequential images taken every 10 seconds for 5 minutes.
Figure 4. Photostability comparison by microscopy. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF™680R, CF™F680, Cy®5.5 or Alexa Fluor® 680 (AF680) goat anti-mouse IgG conjugates. Cells were imaged using an Olympus mercury arc lamp microscope equipped with a Cy®5 filter set and CCD camera. The graph illustrates the relative fluorescence intensities of sequential images taken every 10 seconds for 5 minutes.
Figure 5. HeLa cells were stained with mouse a-tubulin antibody followed by CF™680 goat anti-mouse IgG. Images were captured using an Olympus mercury arc lamp microscope equipped with a Cy®5 filter set, CCD camera and ImagePro Express software.
Figure 5. HeLa cells were stained with mouse a-tubulin antibody followed by CF™680 goat anti-mouse IgG. Images were captured using an Olympus mercury arc lamp microscope equipped with a Cy5 filter set, CCD camera and ImagePro Express software.
Figure 6. Near-IR CF™680 and CF™770 for two-color Western blotting. Two-fold dilutions of HeLa cell lysate were run on an acrylamde gel, transferred to a nitrocellulose membrane and probed with mouse alpha-tubulin and rabbit COX IV primary antibodies followed by goat antimouse CF™770 or IRDye® 800 (green) and goat anti-rabbit CF™680 or IRDye®680 (red) at the same final concentrations. After probing, membranes were dried and scanned using an Odyssey® infrared imaging system (LI-Cor® Biosciences). Quantitation of the bands showed approximately a 3.5-fold higher fluorescence intensity of CF dyes compared to the respective IRDye® secondary antibodies (LI-Cor®).
Figure 6. Near-IR CF™680 and CF™770 for two-color Western blotting. Two-fold dilutions of HeLa cell lysate were run on an acrylamde gel, transferred to a nitrocellulose membrane and probed with mouse alpha-tubulin and rabbit COX IV primary antibodies followed by goat antimouse CF™770 or IRDye® 800 (green) and goat anti-rabbit CF™680 or IRDye®680 (red) at the same final concentrations. After probing, membranes were dried and scanned using an Odyssey® infrared imaging system (LI-Cor® Biosciences). Quantitation of the bands showed approximately a 3.5-fold higher fluorescence intensity of CF dyes compared to the respective IRDye® secondary antibodies (LI-Cor®).