Alexa Fluor® conjugated secondary antibodies

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. 

• 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

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 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.

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.

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 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.

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 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.

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 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.

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.

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:

For optimal performance, use Alexa Fluor® conjugated secondary antibodies without spectral overlap. Common combinations for double and triple immunostaining include: