CF™ Dyes: Enhanced Fluorophores with Novel Dye Chemistry

CF dyes are a series of highly water-soluble fluorescent dyes spanning the visible and near-infrared (near-IR) spectrum (Figures 1A and 1B) for labeling antibodies, proteins, nucleic acids, and other biomolecules. Developed by scientists using new breakthrough chemistries, the brightness, photostability, and color selection of CF dyes rival or exceed the quality of other commercial dyes as a result of rational dye design.

Fluorescent Dye Comparison Chart

Figure 1A

Figure 1B

Achieve greater brightness, photostability, and sensitivity with CF dyes

Sigma currently offers 19 CF dyes spanning the visible and near-IR wavelengths, with additional colors in development. The CF dye product line includes reactive CF dyes, labeling kits, CF-labeled secondary antibodies, and other bioconjugates. This collection further expands Sigma’s broad range of carefully selected secondary antibodies and conjugates, allowing scientists to achieve greater sensitivity, brighter results, and better photostability in immunoassays (Figures 2A, 2B, and 2C).

Figure 2A. Relative fluorescence of CF543 and Alexa Fluor 546 (AF546) goat anti-mouse conjugates as a function of the number of dye molecules per protein (degree of labeling).

Figure 2B. Relative photostability of CF633 and Alexa Fluor 647 (AF647) goat anti-mouse conjugates. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 followed by CF633 or Alexa Fluor 647 goat anti-rabbit IgG conjugates. Cells were imaged using a mercury arc lamp microscope equipped with a Cy5 filter set and CCD camera. Sequential images were captured at 0, 1, and 5 minutes.

Figure 2C. Near-IR CF dyes are highly water soluble without carrying excessive negative charge, which can increase non-specific binding of antibody conjugates. Near-IR western blots imaged using the Odyssey system (Li-COR Biosciences). Two dilutions of HeLa cell lysate were probed with mouse anti-tubulin antibody followed by goat anti-mouse conjugated to Alexa Fluor 790 (AF790) (2, 3) or CF790 (5, 6).

To help researchers evaluate their secondary antibody products with complete peace of mind, Sigma is offering the Bioguarantee on all CF dye labeled antibody products.

 

Fluorescent Dye Comparison Chart

CF™ dye Reactive forms λEx (nm) λEm (nm) MW ε Excitation source Direct replacement for: Reactive forms λEx (nm) λEm (nm) MW ε Advantages of CF™ dye
CF™ 350 amine, aminooxy, hydrazide, maleimide, SE 347 448 496 18,000 UV AMCA NHS, sulfo-NHS, hydrazide, HPDP, SE 353 442 330 19,000 - Yields the brightest blue fluorescent antibody conjugates when excited at ~350 nm
- Highly water-soluble and insensitive to pH
Cascade Blue® Acetyl azide, hydrazid, ethylenediame 400 420 596 28,000
DyLight™ 350 NHS, maleimide 353 432 899 15,000
CF™ 405S amine, aminooxy, maleimide, SE 404 431 1169 33,000 405 nm laser Alexa Fluor® 405 SE, cadaverine 400 424 1028 35,000 - Much brighter than Alexa Fluor® 405 due to better compatibility with excitation and emission windows on common instruments
DyLight™ 405 NHS, maleimide 400 420 793 30,000 - Highly water-soluble and insensitive to pH
CF™ 405M aminooxy, maleimide, SE 408 452 503 41,000 405 nm laser BD Horizon™ V450 Conjugated 404 448     - More photostable than Pacific Blue® dye
- As bright as Pacific Blue® dye in the blue channel
- Less spill-over fluorescence in the green channel
- Highly water-soluble
eFluor® 450 Conjugated 405 450    
Pacific Blue® SE 405 455 406 46,000
CF™ 488A amine, aminooxy, hydrazide, maleimide, SE 490 515 914 70,000 488 nm laser Alexa Fluor® 488 5-SDPE, 5-TFPE, maleimide, alkyne, azide, cadaverine, hydrazide, hydroxylamine, SE 494 517 643 73,000 - Minimal charge compared to Alexa Fluor® 488 reduces nonspecific antibody staining
- Less spill-over fluorescence in the red channel than Alexa Fluor® 488
- Extremely photostable
- Highly water-soluble and pH-insensitive
Cy 2 Bis NHS 489 506 714 150,000
DY-495 NHS, maleimide, carboxylic acid, amino derivative 493 521 623 70,000
DyLight™ 488 NHS, maleimide 493 518 1011 70,000
FAM SE 495 520   83,000
FITC   494 518 289 77,000
Fluorescein NHS, maleimide 490 514 332 93,000
CF™ 543 amine, hydrazide, maleimide, SE 541 560 870 100,000 532 nm or 543, 546 laser Alexa Fluor® 546 N/A 556 573 N/A 112,000 - Significantly brigher than Alexa Fluor® 546
- Highly water-soluble and pH-insensitive
CF™ 555 amine, hydrazide, maleimide, SE 555 565 810 150,000 532 nm or 568 nm laser Alexa Fluor® 555 maleimide, alkyne, azide, cadaverine, hydrazide, SE 555 572 1250 155,000 - Brighter and more photostable than Cy 3
- Minimal charge compared to Alexa Fluor® 555 reduces nonspecific antibody staining
Cy 3 Bis NHS, hydrazide, maleimide, NHS 552 566 767 150,000
DY-547 NHS, maleimide, carboxylic acid, amino derivative 557 574 739 150,000
DyLight™ 550 NHS, maleimide 562 576 1040 150,000
TRITC   557 576 479 100,000
CF™ 568 amine, aminooxy, hydrazide, maleimide, SE 562 583 714 100,000 532 nm or 568 nm laser Alexa Fluor® 568 maleimide, cadaverine, hydrazide, SE 578 602 792 88,000 - Optimal for the 568 nm line of the Ar-Kr mixed-gas laser
- Brighter and more photostable than Alexa Fluor® 568
DY-560 NHS, maleimide, carboxylic acid, amino derivative 559 578 769 120,000
Rhodamine Red NHS 570 590 654 129,000
CF™ 594 amine, aminooxy, hydrazide, maleimide, SE 593 614 729 115,000 532 nm or 594 nm laser Alexa Fluor® 594 maleimide, alkyne, azide, cadaverine, hydrazide, SE 590 617 820 92,000 - Yields the brightest antibody conjugates among spectrally similar dyes
- Extremely photostable
DY-594 NHS, maleimide 589 614   92,000
DyLight™ 594 NHS, maleimide 593 618 1078 80,000
Texas Red® SE 595 615 702 116,000
CF™ 620R maleimide, SE 617 639 728 115,000 633 nm or 635 nm laser LightCycler® Red 640 NHS 625 640 758 - Excellent energy acceptor for FRET
- Highly fluorescent
- Extremely photostable
- Highly water-soluble
CF™ 633 amine, aminooxy, hydrazide, maleimide, SE 630 650 821 100,000 633 nm or 635 nm laser Alexa Fluor® 633 maleimide, hydrazide, SE 621 639 1200 159,000 - Yields the brightest antibody conjugates among spectrally similar dyes Optimal for 633 nm He-Ne laser or the 635 nm red diode laser
- Far more photostable than Alexa Fluor® 647
- Highly water-soluble
DY-630 NHS, maleimide, carboxylic acid, amino derivative 636 657 732 200,000
DyLight™ 633 NHS, maleimide 638 658 1066 170,000
CF™ 640R amine, aminooxy, hydrazide, maleimide, SE 642 662 832 105,000 633 nm, 635 nm, or 640 nm laser Alexa Fluor® 647 maleimide, alkyne, azide, cadaverine, hydrazide, SE 651 672 1250 270,000 - Most photostable among spectrally similar dyes
- Yields highly fluorescent protein conjugates
- Very water-soluble and pH-insensitive
Cy 5 Bis NHS, hydrazide, maleimide, NHS 649 666 792 250,000
CF™ 647 hydrazide, maleimide, SE 650 665 836 240,000 633 nm, 635 nm, or 640 nm laser Alexa Fluor® 647 maleimide, alkyne, azide, cadaverine, hydrazide, SE 651 672 1250 270,000 - Antibody conjugates have the best signal-to-noise ratio compared to Alexa Fluor® 647, DyLight™ 649 and Cy 5
- Highly fluorescent
- Highly water-soluble and pH-insensitive
Cy 5 Bis NHS, hydrazide, maleimide, NHS 649 666 792 250,000
DY-647 NHS, maleimide, carboxylic acid, amino derivative 653 672 762 250,000
DyLight™ 650 NHS, maleimide 652 672 1066 250,000
CF™ 660C maleimide, SE 667 685 3112 200,000 633 nm, 635 nm, or 640 nm laser Alexa Fluor® 660 maleimide, SE 668 698 1100 132,000 - Much brighter than Alexa Fluor® 660
- More photostable than Alexa Fluor® 660
- Highly water-soluble
CF™ 660R aminooxy, maleimide, SE 663 682 888 100,000 633 nm, 635 nm, or 640 nm laser Alexa Fluor® 660 maleimide, SE 668 698 1100 132,000 - Brighter than Alexa Fluor® 660
- The most photostable 660 nm dye, ideal for confocal microscopy
- Highly water-soluble
CF™ 680 maleimide, SE 681 698 3241 210,000 680 nm or 685 nm laser Alexa Fluor® 680 maleimide, SE 684 707 1150 183,000 - The brightest among spectrally similar dyes
- Superior signal-to-noise ratio in imunostaining
- Highly water-soluble and pH-insensitive
Cy 5.5 Bis NHS, hydrazide, maleimide, NHS 675 695 1128 250,000
DyLight™ 680 NHS, maleimide 692 712 950 140,000
IRDye® 680 NHS, maleimide 676 693 1402 250,000
CF™ 680R aminooxy, maleimide, SE 680 701 912 140,000 680 nm or 685 nm laser Alexa Fluor® 680 maleimide, SE 684 707 1150 183,000 - The most photostable 680 nm dye
- Suitable for labeling nucleic acids and small bio-molecules
- Highly water-soluble and pH-insensitive
- CF680/CF770 double labeling compatible with Li-COR Odyssey System
Cy 5.5 Bis NHS, hydrazide, maleimide, NHS 675 695 1128 250,000
DyLight™ 680 NHS, maleimide 692 712 950 140,000
IRDye® 680 NHS, maleimide 676 693 1402 250,000
CF™ 750 SE 755 777 3009 250,000 680 nm, 685 nm, or 785 nm laser Alexa Fluor® 750 maleimide, SE 753 782 1300 290,000 - Exceptionally bright and stable
- Less immunogenic than competing dyes
- Better signal-to-noise ratio compared to APC- Alexa Fluor® 750 tandem dye with 633 nm excitation
Cy 7 Bis NHS, NHS 743 767 818 250,000
DyLight™ 750 NHS, maleimide 752 778 1034 210,000
APC-Alexa Fluor® 750 Conjugated 650 779   240,000
CF™ 770 SE 770 797 3138 220,000 785 nm laser DyLight™ 800 NHS, maleimide 777 794 899 270,000 - Exceptionally bright and stable
- Less immunogenic than competing dyes
- CF680/CF770 double labeling compatible with Li-COR Odyssey System
IRDye® 800CW NHS, maleimide, carboxylate 778 794 1166 300,000
CF™ 790 SE 784 806 3267 210,000 785 nm laser Alexa Fluor® 790 SE 785 810 1750 260,000 - Exceptionally bright and stable
- Less immunogenic than competing dyes

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Materials