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Validated Alexa Fluor® secondaries for guaranteed performance

Related

  • Alexa Fluor® secondary antibodies
    • Alexa Fluor® 488 conjugated secondaries
      • Alexa Fluor® 594 conjugated secondaries
        • Alexa Fluor® 647 conjugated secondaries
          • Compare Alexa Fluor® 488 to FITC
              • Cell imaging
                • Direct vs indirect IF
                  • Fluorochrome chart
                    • Secondary antibody selection guide
                      • ICC protocol
                        • Alexa Fluor® FAQs

                          Learn how our fluorescent secondary antibodies conjugated to Alexa Fluor®, optimized for cell staining, flow cytometry and fluorescent western blot, ensure you get the best results in immunofluorescence. 

                          • Image shows cells staining with anti-Rabbit Alexa Fluor® 488 secondary antibody in green and Alexa Fluor® 594

                          Validation

                          Products have been extensively tested within our own facilities in applications such as cell imaging, flow cytometry and/or fluorescent western blotting. Robust QC procedures guarantee high performance for each individual product.

                          Quality

                          The ratio of dye molecules per secondary antibody has been optimized to minimize photobleaching and ensure bright staining. Furthermore, each secondary conjugate undergoes a column separation step to remove free dye thus ensuring minimal background staining.

                          Specificity

                          We now offer Alexa Fluor® conjugated secondary antibodies against specific IgG subclasses (eg, IgG2a and IgG2b) for the detection of multiple targets to which primary antibodies raised in different species are not available.

                          A large catalog of mouse monoclonal primary antibodies has been developed over the years. The possibility to detect subclass-specific mouse monoclonal antibodies in a reliable way adds enormous flexibility to your multiplex experiments.

                          Customer Abreview

                          "The spectral location in the red made this perfect for a double label with the other fluorophore in the blue... It has stood up well to storage in the refrigerator and gives a good signal after 6 months."

                          Browse Alexa Fluor® secondaries

                          Example data

                          Cell imaging

                          Image : courtesy of Dr. Shaohua Li, UMDNJ-Robert Wood Johnson Medical school.

                          Sample
                          Embryoid bodies (EBs) obtained upon differentiation of mouse embryonic stem cells.

                          Green staining
                          Primary antibody rabbit anti-laminin. Secondary antibody goat anti-rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed (ab150081).

                          Red staining
                          Primary antibody mouse anti-disabled 2. Secondary antibody goat anti-mouse IgG - H&L (Alexa Fluor® 594) pre-adsorbed (ab150120).

                          Blue staining
                          Nuclei counter stained with DAPI.

                          Fluorescent western blot

                          Sample
                          HeLa cell extract - lanes 1 to 5, decreasing amounts of total cell protein, 25µg, 20µg, 15µg, 10µg and 7µg.

                          Red staining
                          Alpha tubulin (primary ab18251 + anti-rabbit Alexa Fluor® 790, ab175773).

                          Green
                          GAPDH (primary ab83956) + anti-chicken Alexa Fluor® 680, ab175787). 

                          Flow cytometry

                          Summary                                                                                                             

                          Overlay histogram showing subclass specificity of rat anti-mouse IgG3 gamma chain. Alexa Fluor® 647 (ab172329) reacts specifically with primary mouse IgG3 (red) but not other mouse IgG subtypes namely IgG1 (black), IgG2a (green), IgG2b (pink) and IgM (light blue).


                          Alexa Fluor® secondaries

                          Alexa Fluor®Color in vial*Absorption Max (nm)Emission Max (nm)Emission Color**Matching Dyes
                          405Colorless402421BlueCascade Blue
                          488Yellow495519GreenCy2, FITC (fluorescein)
                          555Pink555565OrangeCy3, TRITC (Rhodamine)
                          568Violet578603Orange/ RedRhodamine Red
                          594Purple590617RedTexas Red
                          647Blue650668Far RedAPC, Cy5
                          680Blue679702Near-IR***Cy5.5, IR680
                          750Cyan749775Near-IR***Cy7
                          790Cyan784814Near-IR***IR800

                          * Appearance of the solution in the vial

                          ** Typical emission color seen through a conventional fluorescence microscope with appropriate filters

                          *** It is not possible to view the light emitted by near-IF fluorescent dyes since human vision is insensitive to light beyond 650 nm approximately.


                          Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

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