For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online
United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    Proteins and Peptides
    By product type
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex miRNA assays
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Customized Products & Partnerships
    Customized Products & Partnerships

    Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs.

    Customized products

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

Conjugate selection guide for secondary antibodies

Related

  • Secondary antibodies
    • Alexa Fluor® conjugated
      • IRDye® conjugated
        • Biotin conjugated
          • HRP conjugated
            • Pre-absorbed secondary antibodies
                • Fluorescence
                  • Fluorescence guide
                    • Direct vs indirect IF
                      • Compare Alexa Fluor® 488 to FITC
                          • Western blot
                            • Secondaries optimized for chemiluminescence
                              • Infrared fluorescent western blot
                                • WB protocol


                                    Learn about biotin-labeled antibodies, HRP, and fluorescent secondary antibodies so you can choose the correct secondary antibody for your application.

                                    Secondary antibodies can be conjugated to enzymes, biotin, and fluorescent dyes/proteins. The conjugate choice depends upon the experimental application and allows colorimetric, fluorescent, or chemiluminescent detection of primary antibodies in cell imaging, immunohistochemistry, flow cytometry, and western blotting.

                                    See table below what are the commonly used applications for different conjugated secondary antibodies such as biotinylated antibody, HRP,  and Alexa Fluor® fluorescent secondary antibodies.

                                    Immunoassay experimentSecondary antibody label

                                    Immunocytochemistry, IHC-Fr

                                    Alexa Fluor®,  DyLight®

                                    Immunohistochemistry

                                    Biotin , HRP or HRP-polymer

                                    Flow cytometryAlexa Fluor®,  Phycoerythrin, FITC
                                    Western blotEnzymes - HRP or AP
                                    ELISAEnzymes -  HRP  or  biotin
                                    See all secondary antibodies

                                    Biotin/streptavidin

                                    Multiple biotin molecules can be conjugated to a secondary antibody, biotinylated secondary antibody provides an amplification that makes biotin-conjugated secondary antibodies suitable for detecting proteins expressed at low levels. Visualization is through interaction between biotin and streptavidin, where the streptavidin is bound to labels such as HRP or fluorescent probes. During the assay, it is normal procedure for the biotin-labeled secondary antibody to be added first, sequentially followed by the streptavidin conjugated to a label.

                                    • Get Biotin-labeled secondary antibodies
                                    • See how to detect low abundance proteins in immunohistochemistry


                                    Fluorescent labeled secondary antibodies

                                    Fluorescent conjugates are preferred for cell imaging, flow cytometry, and immunohistochemistry frozen samples (IHC-Fr). A single dye is excited at a particular wavelength and emits a photon at another wavelength, detected by a fluorescent microscope. Fluorescent conjugate secondary antibodies allow signal amplification detection of primary antibodies in cells and tissues due to increase number of conjugated secondary antibodies that are able to bind to the primary antibody and are able to be detected by microscopy. 

                                    See advantages of Alexa Fluor® conjugated antibodies


                                    Enzymes

                                    Enzyme labels are visualized with chromogenic reactions whereby a soluble colorless substrate is converted to a water-insoluble colored compound. so enzymes like HRP and AP allow colorimetric or chemiluminescent detection of primary antibodies in application like western blot, immunohistochemistry

                                    Commonly used enzymes:

                                    Horseradish peroxidase (HRP) is visualized by chromogenic reactions such as diaminobenzidine (DAB) or chemiluminescence. HRP is a 44kDa glycoprotein enzyme label and is more stable than alkaline phosphatase.

                                    HRP labeled secondary antibodies 

                                    HRP-polymer secondaries by binding more horseradish peroxidase than standard HRP secondary antibodies allows amplification of signal ideal for those low expressing targets in IHC.

                                    Alkaline phosphatase (AP) is a hydrolase enzyme and its signal is often measured through its colorimetric substrate p-nitrophenyl phosphate (pNPP). Used mostly in western blot.

                                    AP labeled secondary antibodies
                                    ​

                                    How to use fluorescent-labeled secondary antibodies?

                                    Tips for choosing a fluorescent-labeled secondary antibody

                                    • Choose the brightest set of fluorochromes for your instrument.
                                    • Spectral overlap can be minimized by choosing the right fluorescent labels.
                                    • Make sure to combine the brightest label with the protein which has the lowest level of expression, and vice versa.
                                    • When using secondary antibodies, check to make sure all the primary antibodies have been raised in different species to ensure cross labeling via the secondaries is not encountered. Remember, we provide secondary antibodies against different sub-types from within the same species (mouse IgG1, IgG2, IgG2a, IgG3 etc), providing further choice.  Obviously, the corresponding subtype primary must be used.
                                    • Pre-adsorbed secondary antibodies are generally used during multi-color analysis to ensure low cross-species reactivity.
                                    • Sample autofluorescence is sometimes a problem and must be taken into consideration when choosing your secondary conjugates. For instance, liver sections autofluoresce in the red channel, thus when staining this tissue type labels which emit in the red channel should be avoided.


                                    Absorption/emission spectrum and extinction coefficient

                                    See below fluorescent conjugates absorption/emission spectrum and extinction coefficient to plan your multicolor panels.

                                    Label

                                    Absorption Max (nm)

                                    Emission Max (nm)

                                    Alexa Fluor® 405

                                    402

                                    421

                                    Cy2

                                    489

                                    506

                                    Alexa Fluor® 488

                                    495

                                    519

                                    FITC

                                    494

                                    520

                                    Alexa Fluor® 555

                                    555

                                    565

                                    Cy3

                                    550

                                    570

                                    Phycoerythrin

                                    488

                                    575

                                    Rhodamine

                                    544

                                    576

                                    Cy3.5

                                    576

                                    589

                                    Alexa Fluor® 568

                                    578

                                    603

                                    PE/Texas Red®

                                    535

                                    615

                                    Texas Red®

                                    595

                                    615

                                    Alexa Fluor® 594

                                    590

                                    617

                                    APC

                                    650

                                    662

                                    Cy5

                                    643

                                    667

                                    Alexa Fluor® 647

                                    650

                                    668

                                    PE/Cy7

                                    488

                                    670

                                    Cy5.5

                                    675

                                    694

                                    PE/Cy5.5

                                    535

                                    694

                                    Alexa Fluor® 680

                                    679

                                    702

                                    Alexa Fluor® 750

                                    749

                                    775

                                    APC/Cy7

                                    633

                                    776

                                    Alexa Fluor® 790

                                    784

                                    814


                                    Read next advantages of using Alexa Fluor® conjugated antibodies


                                    Alexa Fluor® and Texas Red® are registered trademarks of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.  DyLight® is a registered trademark of Thermo Fisher Scientific Inc and its subsidiaries. 

                                    See all secondary antibodies
                                    Get resources and offers direct to your inbox Sign up
                                    A-Z by research area
                                    • Cancer
                                    • Cardiovascular
                                    • Cell biology
                                    • Developmental biology
                                    • Epigenetics & Nuclear signaling
                                    • Immunology
                                    • Metabolism
                                    • Microbiology
                                    • Neuroscience
                                    • Signal transduction
                                    • Stem cells
                                    A-Z by product type
                                    • Primary antibodies
                                    • Secondary antibodies
                                    • Biochemicals
                                    • Isotype controls
                                    • Flow cytometry multi-color selector
                                    • Kits
                                    • Loading controls
                                    • Lysates
                                    • Peptides
                                    • Proteins
                                    • Slides
                                    • Tags and cell markers
                                    • Tools & Reagents
                                    Help & support
                                    • Support
                                    • Make an Inquiry
                                    • Protocols & troubleshooting
                                    • Placing an order
                                    • RabMAb products
                                    • Biochemical product FAQs
                                    • Training
                                    • Browse by Target
                                    Company
                                    • Corporate site
                                    • Investor relations
                                    • Company news
                                    • Careers
                                    • About us
                                    • Blog
                                    Events
                                    • Tradeshows
                                    • Conferences
                                    International websites
                                    • abcam.cn
                                    • abcam.co.jp

                                    Join with us

                                    • LinkedIn
                                    • facebook
                                    • Twitter
                                    • YouTube
                                    • Terms of sale
                                    • Website terms of use
                                    • Cookie policy
                                    • Privacy policy
                                    • Legal
                                    • Modern slavery statement
                                    © 1998-2023 Abcam plc. All rights reserved.