Conjugate selection guide for 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. Some enzymes, like HRP and AP, allow colorimetric or chemiluminescent detection of primary antibodies in applications like western blot and 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
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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.

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.