• Secondary antibodies are used for indirect detection by binding primary antibodies directed against your target of interest.
• Indirect detection provides signal amplification, which is useful for studying low-expressed proteins.
• Alternatively, direct detection can be achieved with a primary conjugate.

How do I choose a secondary antibody?

1.      Host species: Select a secondary antibody against the host species of the primary antibody.

a.      Primary antibodies are most commonly raised in rabbit or mouse so anti-rabbit and anti-mouse secondary antibodies are most common.

2.      Immunoglobulin (Ig) isotype class or subclass:   Choose a secondary antibody that binds the isotype of the primary antibody.

a.      Primary antibodies are typically IgG isotypes.

b.      anti-IgG secondaries usually bind to the heavy & light chains (H&L).

3.      Conjugate: Select your conjugate based on your antibody application. Commonly used labels include:

a.      Fluorescent dyes (e.g Alexa Fluor®, CyDye®, DyLight®, APC, FITC, PE)

i.      Choose the brightest set of fluorochromes for your instrument.

b.      Enzymes (horseradish peroxidase (HRP) or alkaline phosphatase (AP))

i.      Forms a colored precipitate when combined with the appropriate substrate

c.      Small molecules (e.g. biotin)

i.      Useful for signal amplification

d.      Gold nanoparticles

Things to consider

• Multiplexing. Be careful to ensure that your different secondary antibodies don't cross-react. When using multiple fluorochromes, you must consider spectral overlap. See our comprehensive fluorochrome chart for selection help.
• Pre-adsorbed antibodies. Using a secondary antibody that has been pre-adsorbed with serum from the same species as your sample can prevent cross-reactivity and reduce background.

Fluorescent conjugates

Enzyme conjugates

Other conjugates