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Short answer
In a western blot, the secondary antibody binds the primary antibody and carries the label that produces the signal. It must be raised in a different species from the primary host and directed against that host’s IgG, for example anti-rabbit for a rabbit primary. The label is commonly horseradish peroxidase (HRP) or alkaline phosphatase (AP), or a fluorophore.
Definition and scope
A secondary antibody recognises the primary antibody rather than the target protein. In western blotting, the primary antibody finds the protein on the membrane and the labelled secondary antibody makes that binding visible. This page covers the secondary-antibody decisions that affect a blot: host species, label, cross-adsorption and dilution. For the general relationship between the two antibody types, see primary and secondary antibody; for the full workflow, see western blot protocol basics.
How the secondary antibody is chosen in practice
Match the host species of the primary
Thermo Fisher’s secondary antibody selection guide states that the secondary antibody host should always be different from the primary antibody host, and that a primary derived from a rabbit needs an anti-rabbit secondary. Its western blotting overview adds that an unmodified mouse monoclonal primary needs an anti-mouse IgG (or non-IgG) secondary obtained from a non-mouse host. If the primary carries a tag such as biotin or HA, the secondary is chosen against the tag instead of the species.
Decision checklist
- Record the host species of the primary antibody from its datasheet, and any tag it carries.
- Pick a secondary directed against that host (or tag).
- Confirm the secondary was raised in a different species from the primary host.
- Choose the label (HRP, AP or fluorophore) that matches your detection system.
- Check whether the secondary is cross-adsorbed.
- Treat the dilution as something to determine by experiment.
Types of label: HRP, AP and fluorophore
Thermo Fisher describes horseradish peroxidase and alkaline phosphatase as the two enzymes used most extensively as labels for protein detection. The trade-offs it states are summarised below.
| Label | Stated trade-off |
|---|---|
| Horseradish peroxidase (HRP) | Favoured for high activity rate, good stability, low cost and wide availability of substrates; Thermo Fisher calls it the enzyme of choice for most applications. |
| Alkaline phosphatase (AP) | Reaction rate remains linear, so allowing the reaction to run longer improves sensitivity, but longer reaction times often increase background. |
| Fluorophore | Needs fewer steps because there is no substrate development step; requires special equipment to detect and document the signal; compatible with multiplexing. |
The selection guide frames the HRP question this way: whether the high activity of an HRP conjugate is needed for sensitive detection with a chemiluminescent substrate. How the signal is then captured is covered in film vs CCD vs laser scanner imaging.
Where it matters for labs and supply decisions
Three details belong on a secondary antibody order or specification line, because each follows from the sources above:
- Target host and class. The secondary must be directed against the primary’s host species and IgG, and raised in a different species.
- Conjugate. HRP, AP or a fluorophore, matched to the detection method and equipment available.
- Cross-adsorption. Thermo Fisher says cross-adsorption of secondary antibodies is helpful in eliminating cross-reactivity, and its cross-adsorbed goat anti-mouse IgG example showed very little cross-reactivity and a low background.
Thermo Fisher’s overview says manufacturer-recommended dilutions can range from 1/100 to 1/500,000 from a 1 mg/mL stock solution, and the optimal dilution of a given antibody with a particular detection system must be determined experimentally. A recommended dilution is therefore a starting point rather than a fixed value. Primary antibody choice is a related decision: see polyclonal vs monoclonal antibodies.
Common misunderstandings
“Any anti-IgG secondary will work.”
It has to be directed against the host species of the primary. An anti-mouse secondary will not detect a rabbit primary.
“The secondary can be raised in the same species as the primary.”
The selection guide says the secondary host should always differ from the primary host.
“A longer incubation always gives a better signal.”
With AP, the linear reaction rate means a longer reaction improves sensitivity, but longer reaction times often increase background too. Other causes of a blank blot are listed in the western blot no-bands decision tree. Background from the blocking step is covered in BSA vs milk blocking buffer.
FAQ
Can the secondary come from the same species as the primary?
No. The secondary antibody host should always be different from the primary antibody host. For a mouse monoclonal primary, the secondary is anti-mouse IgG (or non-IgG) from a non-mouse host.
Why is HRP the usual western blot label?
Thermo Fisher lists its high activity rate, good stability, low cost and wide availability of substrates, and calls HRP the enzyme of choice for most applications. AP is the other widely used enzyme label.
What does cross-adsorbed mean?
Cross-adsorption is a processing step for secondary antibodies that helps eliminate cross-reactivity; Thermo Fisher’s example of a cross-adsorbed secondary showed very little cross-reactivity and a low background.
What dilution should I use?
Manufacturers may recommend dilutions from 1/100 to 1/500,000 from a 1 mg/mL stock solution, but the optimal dilution must be determined experimentally for each antibody and detection system.
Sources
- Thermo Fisher Scientific, Overview of western blotting, checked 2026-10-29.
- Thermo Fisher Scientific, Secondary antibody selection guide, checked 2026-10-29.
