All tags Secondary Antibodies Conjugate selection guide for secondary antibodies

Conjugate selection guide for secondary antibodies

A guide to choosing the correct conjugated secondary antibody for your application.

Secondary antibodies can be conjugated to a large number of labels, including enzymes, biotin, and fluorescent dyes/proteins. The label of choice depends upon the experimental application.

Immunoassay experimentSecondary antibody label
ELISAEnzymes (usually HRP or AP), biotin
ImmunofluorescenceFluoresecent label, biotin
ImmunohistochemistryEnzymes, biotin
Flow cytometryFluorescent label, biotin
ImmunocytochemistryFluorescent label, biotin


Enzymes

Enzyme labels are visualized with chromogenic reactions whereby a soluble colorless substrate is converted to a water-insoluble colored compound.

Commonly used enzymes:

Horse radish 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 

Alkaline phosphatase (AP) is a hydroylase enzyme and its signal is often measured through its colorimetric substrate p-nitrophenyl phosphate (pNPP). 

AP labeled secondary antibodies

Biotin/streptavidin

Multiple biotin molecules can be conjugated to a secondary antibody, providing an amplification step that makes this conjugate 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.

Biotin labeled secondary antibodies.

Fluorescent labeled secondary antibodies

Fluorescent conjugates are preferred for multi-color analysis. A single dye is excited at a particular wavelength and emits a photon at another wavelength. Alternatively, a tandem dye consists of a donor and acceptor fluorochrome placed in close proximity, allowing energy transfer between the two.  The tandem dye is excited at the excitation wavelength of the donor molecule and emits a photon at the emission wavelength of the acceptor molecule.

Find out about Alexa Fluor® conjugates for multi-color analysis.

Tips for choosing a fluorescent labeled secondary antibody

  • Choose the brightest set of fluorochromes for your instrument.
  • Spectral overlap can be minimized through 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 take in to 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.


The table below lists fluorescent labeled secondary antibodies within our portfolio.

LabelAbsorption Max (nm)Emission Max (nm)Extinction Coefficient
Alexa Fluor® 40540242135,000
Cy2489506150,000
Alexa Fluor® 48849551973,000
FITC49452070,000
Alexa Fluor® 555555565155,000
Cy3550570150,000
Phycoerythrin4885751,960,000
Rhodamine54457680,000
Cy3.5576589150,000
Alexa Fluor® 56857860388,000
PE/Texas Red®535615-
Texas Red®59561585,000
Alexa Fluor® 59459061792,000
APC650662700,000
Cy5643667250,000
Alexa Fluor® 647650668270,000
PE/Cy7488670-
Cy5.5675694190,000
PE/Cy5.5535694-
Alexa Fluor® 680679702183,000
Alexa Fluor® 750749775290,000
APC/Cy7633776-
Alexa Fluor® 790784814260,000

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.