Alexa Fluor® conjugated secondaries are the brightest fluorescent secondary antibodies, created from Alexa Fluor® dyes. We offer secondary antibodies conjugated to nine Alexa Fluor® dyes that cover the whole spectrum with minimal spectral overlap, thus ideal for multi-color analysis and use in cell imaging and flow cytometry experiments.
Last edited
Fri 28 Apr 2023
Alexa Fluor® conjugated secondary antibodies at abcam
Our range spans over 200 different Alexa Fluor® conjugated secondary antibodies, raised across many species, including donkey, goat, and rabbit. They can be used against specific IgG subclasses (eg IgG2a and IgG2b) to detect multiple targets to which primary antibodies raised in different species are unavailable. To further minimize background from cross-species reactivity, we recommend using Alexa Fluor® pre-adsorbed secondary antibodies.
All products have been extensively tested within our facilities in applications such as cell imaging, flow cytometry, and fluorescent western blot with robust QC procedures.
Advantages of Alexa Fluor® conjugated secondary antibodies
- Brightest dyes: the fluorescence intensity of Alexa Fluor® dyes outperforms other spectrally similar dyes.
- High photostability: allows more time for observation and image capture.
- pH insensitivity: fluorescence intensity remains high over a broad range from pH 4.0-10.0 (optimal pH 6.5-8.5)
- Good water solubility: prevents precipitation and aggregation of secondary antibodies.
- High quality: ratio of dye molecules per secondary is optimized to minimize photobleaching and ensure bright staining.
- Minimal background staining: each secondary conjugate undergoes a column separation step to remove the free dye
Our full range of Alexa Fluor® conjugated secondary antibodies
See below for the Alexa Fluor® dyes we currently offer and their characteristics, including emission and absorption spectra that match the most common operational wavelengths of excitation sources.
Alexa Fluor® 405
Alexa Fluor® 405 is a blue dye, with maximum absorption of 401nm and maximum emission of 421nm. This dye is colorless in the vial, closely matching the fluorescence of the alternative Cascade Blue.
Alexa Fluor® 488
Showing as yellow in the vial, Alexa Fluor® 488 is a green dye that matches the fluorescence of Cy2 and FITC. Excitation is ideally suited to 496 nm for argon ion lasers, with maximum absorption of 495 nm and maximum emission of 519nm. The quantum yield is usually 0.92.
The ideal emission filter for 488 in confocal microscopy is either FITC or Alexa Fluor® 488, and for flow cytometry is 525/30 BP. Alexa Fluor® 488 is most compatible with DAPI, Alexa Fluor® 594, and Alexa Fluor® 647 for any multiple staining.
Example data with AlexaFluor® staining in cell imaging (left): Embryoid bodies from mouse embryonic stem cells stained with Goat Anti-rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed (green), Goat Anti-Mouse IgG - H&L (Alexa Fluor® 594) pre-adsorbed (red) and nuclei counterstained with DAPI (blue). Courtesy of Dr. Shaohua Li, Robert Wood Johnson Medical School.
Alexa Fluor® 555
Alexa Fluor® 555 is an orange dye, showing as a pink solution in the vial, matching Cy3 and TRITC (Rhodamine). This dye has a maximum absorption of 555nm, a maximum emission of 565nm, and a quantum yield of around 0.1.
Alexa Fluor® 568
Alexa Fluor® 568 is a red/orange dye, showing as violet in the vial, matching Rhodamine Red in color. This dye has a maximum absorption of 578nm, a maximum emission of 603nm, and a quantum yield of around 0.69.
Alexa Fluor® 594
Just along the fluorescence spectrum from 568, Alexa Fluor® 594 is also red dye, showing as purple in the vial, matching Texas Red in fluorescence. This dye has a maximum absorption of 590nm, a maximum emission of 617nm, and a quantum yield of around 0.66.
Alexa Fluor® 647
Alexa Fluor® 647 is a far-red dye, showing blue in the vial, matching the fluorescence of APC and Cy5. This dye has a maximum absorption of 650nm, a maximum emission of 665nm, and a quantum yield of around 0.33.
Example of AlexaFluor® performance in flow cytometry (left): Histogram on subclass specificity rat anti-mouse IgG3 gamma chain. Anti-Mouse IgG3 gamma chain (Alexa Fluor® 647) (ab172329) reacts specifically with primary mouse IgG3 (red) but not IgG1 (black), IgG2a (green), IgG2b (pink) and IgM (light blue) subtypes.
Alexa Fluor® 680
Alexa Fluor® 680 is a near-infrared dye, showing as blue in the vial, matching Cy5.5 and IR680 in fluorescence. This dye has a maximum absorption of 679nm, a maximum emission of 702nm, and a quantum yield of around 0.36.
Alexa Fluor® 750
Alexa Fluor® 750 is a near-infrared dye, showing cyan in the vial, matching Cy7 in fluorescence. This dye has a maximum absorption of 749nm, a maximum emission of 775nm, and a quantum yield of around 0.12.
Alexa Fluor® 790
Like 750, Alexa Fluor® 790 is a near-infrared dye, also showing cyan in the vial, but matching IR800 in fluorescence. This dye has a maximum absorption of 784nm and a maximum emission of 815nm.
Example of AlexaFluor® performance in western blot (left): Western blot with HeLa cell extract shows decreasing amounts of total cell protein across 25µg, 20µg, 15µg, 10µg and 7µg. Red staining is alpha tubulin, ab18251 and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 680) (ab175773). Green staining is GADPH, ab83956 and Goat Anti-Chicken IgY H&L (Alexa Fluor® 790) (ab175787).
Tips for multicolor staining
When working with more than one Alexa Fluor® conjugated secondary antibody, it is important to use antibodies with distinct emission maximum (at least 40 nm difference). The emission spectrum for each dye can be seen below:
Image 1 (above): Alexa Fluor® dye spectra by wavelength and fluorescence emission
For optimal performance, use Alexa Fluor®conjugated secondary antibodies without spectral overlap. Common combinations for double and triple immunostaining include:
Immunostaining | Antibody combination |
Double | Alexa Fluor®488 and Alexa Fluor®647 |
Triple | Alexa Fluor®405, Alexa Fluor®555 and Alexa Fluor®647 |
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.
