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Fluorescent dyes for secondary antibodies

Fluorophores, coupled to antibodies or streptavidin, allow analytes to be visualized quickly and easily. There is a large number of fluorescent dyes, each with its own specific properties. In this FAQ you can find out more about Jackson ImmunoResearch secondary antibody conjugates, which can be used in all common immunoassays (including flow cytometry, microscopy, Western blot and ELISA). Choosing the “right” fluorescent dye depends on a number of factors, here are some more tips. The one listed here alphabetical list of fluorophores only lists the dyes that are used by us or our partners for the production of antibody conjugates and does not claim to be complete.

Alexa Fluor® dyes

Alexa Fluor® dyes produce conjugates with a high luminosity and photostability that is superior to most conventional fluorescent dyes. Due to the high absorption capacity in the wavelength range of common excitation sources and a low tendency towards fluorescence quenching ("quenching") after coupling to proteins, bioconjugates of Alexa Fluor dyes produce an exceptionally bright and stable fluorescence. The fluorescence intensity is also insensitive to fluctuations in the pH value (pH 4 - 10). Good water solubility of the dyes makes the conjugates resistant to aggregation and precipitation, which prevents common problems with background staining. Alexa Fluor dyes are closely related to the DyLight dyes due to structural similarities.

Alexa Fluor® 488

Alexa Fluor 488 (Amax 493 nm, Emax 519 nm) has spectral properties comparable to fluorescein (FITC) with regard to excitation and emission, but produces conjugates that are far more fluorescence-intensive, more photostable and less sensitive to pH fluctuations are between pH 4 and 10. Alexa Fluor 488 conjugates are compatible with standard equipment, settings or filters for fluorescein and are ideally suited for all fluorescence microscopic and flow cytometric applications with the highest demands on sensitivity. Alexa Fluor 488 can be made visible longer in the epifluorescence microscope and appear brighter than fluorescein, even without adding anti-fading reagents to aqueous mounting media. Alexa Fluor 488 is also more luminous and long-term stable than fluorescein in permanent organic mounting media. Alexa Fluor 488 is comparable to DyLight 488.

Alexa Fluor® 594

Alexa Fluor 594 Conjugates (Amax 591 nm, Emax 614 nm) fluoresce in the red area of ​​the light spectrum, are more luminous than other red fluorescent dye conjugates and provide a clear one better color separation of green fluorescent dyes as Cy3 or TRITC. Therefore, they are the best choice for immunofluorescence detection in the darker red region of the visible spectrum. Alexa Fluor 594 conjugates fluoresce much more strongly and stably than Texas Red conjugates, are more water-soluble and cause less background coloring. The However, photo stability also depends on the mounting medium: while Alexa Fluor 594 conjugates in ProLong® Gold Antifade (Life Technologies) are more stable than Texas Red conjugates, they fade faster in VECTASHIELD® (Vector Laboratories).

Alexa Fluor® 647

Alexa Fluor 647 conjugates (Amax 651 nm, Emax 667 nm) correspond in their spectral properties almost to those of Cy5 conjugates, but are much brighter than these. If fluorescent secondary antibodies in the near infrared range are to be used, Alexa Fluor 647 conjugates are the best choice for the confocal laser scanning microscopy and a comparable or even better alternative to APC conjugates for flow cytometry. Alexa Fluor 647 conjugates can be combined with many other fluorescent dyes due to the large distance between their emission and other fluorochromes that emit at shorter wavelengths and are ideal for multiple markings in confocal laser microscopy. A particular advantage of Alexa Fluor 647 over other fluorescent dyes is that low autofluorescence of biological samples resulting from excitation with red light. Since the fluorescence at 670 nm cannot be sufficiently perceived by the human eye, Alexa Fluor 647 cannot be made optimally or hardly visible with a conventional epifluorescence microscope, especially since the mercury lamp, which is used in most devices, does not have enough light within the excitation range issued by Alexa Fluor 647. Alexa Fluor 647 conjugates are therefore not recommended for use in epifluorescence microscopy. They are usually made visible with a suitable excitation laser (helium-neon laser (at 633 nm), krypton-ion laser (at 647 nm), diode laser) and an infrared detector. For flow cytometry, Alexa Fluor 647 conjugates are an inexpensive alternative to APC conjugates that are comparable or better in terms of their fluorescence intensity. Due to the smaller molecule size of Alexa Fluor 647 (1.3 kDa) compared to APC (104 kDa), the The resulting antibody conjugates from Alexa Fluor 647 are also much smaller and better suitable for intracellular markings in flow cytometry.

AMCA (aminomethylcoumarin acetate)

AMCA conjugates Due to their spectral properties (Amax 350 nm, Emax 450 nm), they are mostly used for multiple markings both in immunofluorescence microscopy and in flow cytometry. They show little fluorescence overlap with green fluorescent dyes and hardly any or none at all with fluorochromes emitting wavy for longer periods. However, all fluorochromes that are excited in the blue range tend to bleach quickly. Because of this critical optical property, AMCA is not recommended for single markings. If this blue fluorescent dye is used for multiple markings in fluorescence microscopy, it should be used to detect the most strongly expressed antigen, since the human eye perceives this color less than other colors. AMCA can be excited with a mercury lamp and the fluorescence can be observed using a UV filter set. Possibilities to improve the visibility of AMCA include the dark adaptation of the eyes, the use of fluorite instead of glass lenses, the avoidance of UV-absorbing mounting media (e.g. plastic-based media) and the evaluation with blue-light-sensitive sensors. Because of the unfavorable bleaching properties of AMCA in confocal and conventional fluorescence microscopy, the use of antifading reagents such as ImmunonoSelect® Anti-Fading or mounting media with the addition of anti-fading reagents such as e. B. n-propyl gallate (2% n-propyl gallate 80-90% glycerol in PBS (pH 7.2-9.0)). In flow cytometry, AMCA can be excited with a mercury vapor lamp or a water-cooled argon ion laser with emission wavelengths in the UV range. Because of the relatively weak signal and rapid fading, AMCA is not recommended for single-photon-induced fluorescence microscopy. However, AMCA has proven to be signal-intensive and photostable for two-photon microscopy.

Brilliant Violet ™ BD

Brilliant Violet Conjugates BV421 ™ and BV480 ™ offer more options in the blue color channel for multiple markings. This allows up to 5 fluorescent dyes to be used.

Brilliant Violet 421 ™ (BV421 ™)

Brilliant Violet 421 ™ conjugates (Amax 407 nm, Emax 421 nm) are particularly suitable for multiple marking in combination with BV 480 ™, Alexa Fluor® 488, Rhodamine Red ™ -X, and Alexa Fluor® 657. Attention, due to their physical properties, Brilliant Violet 421 ™ also tend to be -Conjugates that are stimulated in the blue range to bleach faster than dyes with higher-wave stimulation!

Brilliant Violet 480 ™ (BV480 ™)

Brilliant Violet 480 ™ conjugates (Amax 436 nm, Emax 478 nm) are particularly suitable for multiple marking in combination with BV 421 ™, Alexa Fluor® 488, Rhodamine Red ™ -X, and Alexa Fluor® 657.

Find out more about BD Brilliant Violet ™ secondary antibody conjugates here.

Cyanine (Cy ™) dyes

Cyanine (Cy ™) dyes belong to a second generation fluorochrome family. you are more photostable and more luminous than, for example, FITC or TRITC. While Cy3 conjugates were widely available due to their special luminosity and photostability, the manufacturer Jackson ImmunoResearch had discontinued the production of Cy2 and Cy5 conjugates and replaced them with the more luminous dyes Alexa Fluor®. In various laboratories it has now been shown that in general all cyanines lead to better staining results when using organic mounting media and the associated strong dehydration during the embedding process. Jackson ImmunoResearch has therefore reintroduced selected Cy2 and Cy5 conjugates, conjugated streptavidin and conjugated IgG controls - especially for multiple labeling - to the program.

Cy2 ™ (carbocyanine)

Cy2 ™ (carbocyanine) (Amax 492 nm, Emax 510 nm) has similar spectral properties as FITC and can be displayed with the same filter sets. When using antifading reagents, it should be noted that it sensitive to N-phenylenediamine is found in some commercial anti-fading media.

Cy2 conjugate Cy3 ™ (indocarbocyanine)

Cy3 ™ (indocarbocyanine) (Amax 550 nm, Emax 570 nm) is more luminous, more photostable and shows less background than TRITC and most other fluorescent dyes. Cy3 conjugates can without additional equipment can be used with a conventional TRITC filter set, since the excitation and emission spectra are almost identical to those of tetramethylrhodamine (TRITC). Special Cy3 filters (e.g. Zeiss filter set 20) are also available from various suppliers. Cy3 can be excited to 50% of its maximum absorption with the 514 nm or 528 nm rays of an argon laser, to 75% of the maximum with a helium-neon laser (at 543 nm) or with a mercury lamp (at 546 nm) . Cy3 is often used with a dye that fluoresces in the green spectrum for double marking. Because of the spectral overlap of the fluorescence emission, the use of a narrow bandpass filter for the green channel is recommended to minimize the Cy3 fluorescence in the filter set of the green dye. If a confocal microscope with a krypton-argon laser and an infrared detector (CCD) is available, Cy3 can also be combined with dyes in the near infrared range such as Alexa Fluor 647 for multiple markings.

Cy5 ™ (indodicarbocyanine)

Cy5 ™ (indodicarbocyanine) (Amax 650 nm, Emax 670 nm) is suitable because of its minimal overlap with other fluorochromes especially for multiple markings. Since the fluorescence of Cy5 cannot be sufficiently perceived with the human eye, Cy5 cannot be made optimally or hardly visible with a conventional epifluorescence microscope, especially since the mercury lamp, which is used in most devices, does not have enough light within the excitation range of Cy5 emitted. The dye is therefore particularly suitable for use in confocal laser scanning microscopy. Cy5 can be excited by a krypton / argon laser (98% of the maximum absorption with 647 nm wavelength), a helium / neon laser (63% of the maximum absorption with 633 nm wavelength) or with a red diode laser.

DyLight ™ dyes

DyLight ™ dyes belong to a family of fluorochromes comparable to Alexa dyes with improved brightness and photo stability. They are highly soluble in water and retain their luminosity in a pH range from 4 to 9.

DyLight ™ 405

DyLight 405 conjugated antibodies (Amax 400nm, Emax 421nm) are very bright and photostable, but their use on confocal microscopes with a 405 nm laser and suitable emission filter. Here DyLight 405 can work in both aqueous and permanent mounting media with high color separation and sensitivity can be used effectively for four-color imaging in the blue area. The best color separation can be achieved in combination with Alexa 488, Rhodamin Red-X and Alexa 647. With a slightly lower color separation, the use of Cy3 instead of Rhodamine Red-X is also possible. When using aqueous mounting media, we recommend adding n-propyl gallate to prevent the fluorescence from fading (mounting medium e.g. n-propyl gallate-glycerol). As with all fluorophores that are excited in the blue range, the photostability is lower compared to higher-wave excitation. The use of DyLight 405 in conventional fluorescence microscopy and in flow cytometry is not recommended, since the optimal filter sets / excitation sources are usually not available here.

More DyLight antibody conjugates can be found here.

FITC (fluorescein isothiocyanate)

FITC (Amax 492 nm, Emax 520 nm) is a fluorescein derivative which, due to its long history, is still widely used today. FITC is the form of fluorescein that is linked to all Jackson ImmunoResearch Labs antibodies and proteins except streptavidin. This is one of the most serious drawbacks rapid fading (Fading) and the relative to newer fluorophores low luminosity. Because of the unfavorable bleaching properties, the addition of antifading reagents is recommended when using FITC-conjugated antibodies. Alternatives with better fading properties with the same excitation are therefore Cy2 ™ (carbocyanine) or Alexa Fluor® 488. DTAF (dichlorotriazinylamino-fluorescein) and FITC (fluorescein isothiocyanate) are derivatives of the same fluorescein molecule, which is why DATF has identical excitation and emission maxima as FITC . For coupling to streptavidin, Jackson ImmunoResearch Labs uses DTAF instead of FITC, since the fluorescence of FITC is extinguished considerably after coupling to streptavidin (effect of fluorescence quenching / quenching effect). This phenomenon is unique to streptavidin and is not observed when coupled to antibodies. For streptavidin conjugates too, alternatives with better fading properties are the dyes Cy2 ™ (carbocyanine) or Alexa Fluor® 488.

TRITC, Rhodamin Red ™ -X and Texas Red®

TRITC, Rhodamine Red-X, and Texas Red are rhodamine derivatives that fluoresce in the orange or red area of ​​the visible spectrum. Conjugates of these rhodamine dyes each have different excitation maxima (550, 570 and 596 nm) and emission maxima (570, 590 and 620 nm). All three fluorochromes are for Suitable for double markings in combination with FITC or Alexa Fluor 488. Although TRITC has traditionally been combined with FITC in double markings, better color separation can be achieved with Rhodamine Red-X or Texas Red because of the smaller overlap with the FITC spectrum. As less background, brighter and more stable As an alternative to Texas Red, the use of Alexa Fluor 594 conjugates is recommended. Cy3 is also a more luminous alternative to TRITC. Phycoerythrin (instead of rhodamine) is recommended for double labeling with FITC in flow cytometry, since both fluorophores can be excited with an argon laser (488 nm).

Rhodamine Red ™ -X (RPX)

Rhodamine Red ™ -X (Rhod. Red-X, RRX) is one because of its spectral properties almost ideal fluorochrome for triple and quadruple markings with DyLight 405, Alexa Fluor 488, and Alexa Fluor 647 when using a confocal laser microscope equipped with a 405 nm laser and a krypton-argon laser. The fluorescence emission of Rhodamine Red-X lies roughly in the middle between that of Alexa Fluor 488 and Alexa Fluor 647 and shows little overlap with these dyes. In addition, all three fluorochromes can be detected by the krypton-argon laser, which is usually present in confocal laser microscopes, without any relevant loss of sensitivity, since the emitted laser beams at 488 nm, 568 nm and 647 nm wavelength are each the optimal excitation wavelength for Alexa Fluor 488 (FITC), rhodamine Red-X and Alexa Fluor 647 represent. By switching on a 405 nm laser, four-color marking with DyLight 405-conjugated secondary antibodies is possible. In conventional fluorescence microscopy, Rhodamine Red-X conjugates are particularly suitable for combining with green fluorescent dyes (Alexa Fluor 488). The simultaneous use of Rhodamine Red-X -coupled antibodies with blue fluorescent AMCA conjugates for double labeling is also possible.

R-Phycoerythrin (R-PE), Allophycocyanin (APC) and Peridinin-Chlorophyll (PerCP)

R-PE (240 kDa), APC (104 kDa) and PerCP (35 kDa) belong to the light-harvesting phycobiliproteins, a family of naturally occurring, fluorescent macromolecules from the photosynthesis systems of various algae. Since phycobiliproteins have more than 30 covalently bound tetrapyrrole pigments, so-called phytochromobilins, the fluorescence signals are particularly intense. In the photosynthesis systems of algae, these molecules absorb and transmit light (energy) to the chlorophyll of the photosynthetic reaction centers by means of fluorescence resonance energy transfer (FRET).The arrangement of the pigments in the protein is naturally optimized in such a way that maximum light collection and fluorescence can be achieved with minimal fluorescence quenching due to internal or external environmental influences. After conjugation of the phycobiliproteins to the highly purified secondary antibodies, the fluorescence quenching is very low in contrast to conventional fluorophore-antibody conjugations. Antibody conjugates with phycobiliproteins have a very high specific fluorescence activity with high extinction coefficients and thus a very high quantum yield. The fluorescence of some phycobiliproteins corresponds roughly to that of 30 fluorescein or 100 rhodamine molecules with a comparable excitation. Phycobiliprotein conjugates can be excited over a wide range of wavelengths and are characterized by large Stokes shifts of their emission maxima into longer-wave ranges. Other advantages of antibody conjugates with phycobiliproteins are theirs high stability, one good solubility in water and a high pH insensitivity of fluorescence. These properties, together with the high fluorescence intensity, have proven particularly useful in techniques based on laser excitation such as flow cytometry. PerCP, Alexa Fluor 488 (or FITC) and R-PE are excited with the 488 nm main line of an argon laser and can thus be used for single, double or triple staining with single laser flow cytometers. APC or Alexa Fluor 647 can be used as the fourth color by excitation at 633 or 635 nm with a double laser flow cytometer. The relatively high molecular weights of R-PE, APC and PerCP have limited use in procedures that require good cell and tissue penetration. They are therefore mainly suitable for marking the surface of cells in flow cytometry. R-PE and APC can be excited by light over a wide range of the visible spectrum, are highly water soluble, have relatively low isoelectric points, and are free of potentially sticky carbohydrates. They are particularly suitable for applications that require either high sensitivity, good color separation or multiple marking.

R-phycoerythrin (R-PE)

The 240 kDa phycobiliprotein R-PE is isolated from macrophytic algae (seaweed). The absorption maximum is at a wavelength of 490 nm, with a smaller adsorption peak at 545/566 nm and emits light with a maximum fluorescence at 580 nm.

Allophycocyanin (APC)

APC is a phycobiliprotein isolated from blue-green Spriulinia algae, which is chemically cross-linked for stabilization. It contains the chromophoric group phycocyanobilin, with an absorption maximum at 650nm and a maximum emission at 660nm.

Peridinin Chlorophyll Protein (PerCP)

PerCP ist a peridinin-chlorophyll complex of approx. 35.5 kDa obtained from dinoflagellates (Dinophyceae sp.). It has a broad excitation spectrum with a main absorption peak at 482 nm, a second absorption peak at 442 nm and a long Stokes shift to an emission maximum at 677 nm.

Alexa Fluor®, Rhodamin RedTM-X, and Texas Red® are registered trademarks of Life Technologies, Inc. *. Cy ™ is a trademark of GE Healthcare (patent 5,268,486). Brilliant Violet ™ is a trademark of Sirigen Inc., a Becton, Dickinson and Company affiliate. DyLight ™ is a trademark of Thermo Fisher Scientific. For all dyes and their dye products, Jackson ImmunoResearch Labs has license agreements with the aforementioned companies for the dye technologies with regard to the use, manufacture, trade and sale.

All images Jackson ImmunoResearch.