All tags Secondary Antibodies Conjugate selection guide for secondary antibodies

Conjugate selection guide for secondary antibodies

by Brian Carpenter

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

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

Immunoassay experimentSecondary Antibody Label
ELISAEnzymes (usually HRP or AP), biotin
ImmunofluorescenceFluoresecent label, biotin
ImmunohistochemistryEnzymes, biotin
Flow CytometryFluorescent label, biotin
ImmunocytochemistryFluorescent label, biotin

Enzymes - are proteins, and the molecules upon which they act are known as substrates.  The enzyme label can be visualized by means of enzyme histochemical methods via chromogenic producing color reactions, essentially a soluble colorless substrate is converted to a water-insoluble colored compound.

The two most used enzymes are:

Horse radish peroxidase (HRP) - can be 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 pNNP. 

AP labeled secondary antibodies

Biotin/Streptavidin - is commonly used when the target of interest is expressed at low levels and cannot be detected using secondary labeled antibodies alone.  Many biotin molecules can be conjugated to a secondary antibody with the additional advantage of streptavidin having a high affinity for biotin.  Through this amplification step and having the streptavidin bound to labels such as HRP or fluorescent probes, proteins which are expressed at low levels are more likely to be detected.  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 e.g. HRP.

Biotin labeled secondary antibodies.

Fluorescently conjugated secondaries - for multi-color analysis there is a preference for scientists to use secondary antibodies conjugated to fluorescent labels. Due to their novel electronic configurations, fluorochromes have a unique and characteristic spectra for absorption and emission - 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 molecules being placed in close proximity, allowing for energy transfer between the two.  The tandem dye is excited at the excitation wavelength of the acceptor molecule and emits a photon at the emission wavelength of the donor molecule.

Of special note - we currently offer a range of Alexa fluor® conjugates which are ideal for multi-color analysis that span the whole of the spectrum from the UV to far infra-red.  Furthermore, the Alexa fluor® dyes are conjugated to secondary antibodies against a diverse set of species, plus a large number are pre-adsorbed ensuring low cross-reactivity.

Considerations for choosing a fluorescent conjugated secondary antibody

The table below is a list of fluorescently labeled secondary antibodies within our portfolio.  For convenience, we also have provided the absorption max, emission max and extinction coefficient.

Tips for choosing a fluorescently labeled secondary for multi-color analysis:

  • 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. 

LabelAbsorption Max (nm)Emission Max (nm)Extinction Coefficient
Alexa Fluor® 40540242135,000
DyLight® 48849351870,000
Alexa Fluor® 48849551973,000
Alexa Fluor® 555555565155,000
DyLight® 550562576150,000
Alexa Fluor® 56857860388,000
PE/Texas Red®535615-
Texas Red®59561585,000
Alexa Fluor® 59459061792,000
DyLight® 59459365880,000
Alexa Fluor® 647650668270,000
DyLight® 650652672250,000
Alexa Fluor® 680679702183,000
Alexa Fluor® 750749775290,000
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.