Western blot secondary antibody selection: choosing a primary antibody's matching secondary antibody western blot reagent by host species, why choosing secondary antibody comes after deciding which primary antibodies and which primary antibody western blot or western blot primary antibody pairing the target allows, when an anti mouse secondary antibody, an anti rabbit secondary antibody, an anti rabbit antibody or a secondary antibody for western blot raised against another species is the one you need, what an hrp secondary antibody conjugate, a secondary antibody hrp format, western blot hrp detection, an anti hrp antibody control, an anti rabbit hrp secondary antibody and an anti mouse hrp secondary antibody give that fluorescent secondary antibodies and an anti rabbit fluorescent secondary antibody do not, why a detection antibody, a capture antibody and a capture and detection antibody pair are different products, and how cross-adsorption stops secondary antibodies for western blot work colliding

The rules, in order

1. Match the host of the primary, not the sample. A secondary is described as anti-something raised in something: an anti-rabbit raised in goat detects a rabbit primary. Read it as a sentence and the confusion disappears. The secondary must recognise the primary's host species, and it must not be raised in the same species as your sample.
2. Cross-adsorption where species could collide. Cross-adsorbed secondaries have been passed over immobilised serum from other species to remove antibodies that would bind them. Use them for multiplexing, for samples from a species close to the secondary's host, and any time an unexplained band appears with the secondary alone.
3. Enzymatic or fluorescent label. Horseradish peroxidase with chemiluminescence is sensitive and has a limited dynamic range that saturates easily, which makes quantitation awkward. Fluorescent secondaries give a wider linear range and straightforward two-colour multiplexing, and need an imager. For quantitative work the fluorescent route is usually the better answer.
4. Titrate it, like the primary. The dilution printed on the bottle is a starting point. Running a small dilution series on your own blot finds the concentration giving the best signal to background, saves reagent and removes most background problems at a stroke.
5. Run the secondary-only control. Incubate a blot with the secondary and no primary. Any band that appears is the secondary binding something directly, and knowing that before you interpret a result saves a great deal of argument later.

Fragments, conjugates and special cases

Fragment secondaries reduce non-specific binding to receptors on some sample types and are worth knowing about when a blot of immune tissue misbehaves. Protein A and protein G conjugates bind many species' antibodies and are a fallback when no suitable species-specific secondary exists.

For detecting a primary raised in the same species as the sample, specialised reagents exist that bind the primary preferentially. They are more expensive and are the honest answer to a problem that otherwise has none.

Storage and lot behaviour

Conjugated secondaries degrade with light exposure and freeze and thaw cycles, and a secondary that has quietly lost activity presents as a failing primary. Aliquot on arrival, keep fluorescent conjugates dark, and follow the supplier's storage instruction rather than the freezer convention.

Keep a reference blot image from a known-good lot. Comparing against it is the fastest way to tell whether a new lot or the sample changed.

Common questions

How do I choose a western blot secondary antibody?

It must be raised against the host species of your primary and must not be raised in the same species as your sample. Read the name as a sentence: an anti-rabbit raised in goat detects a rabbit primary.

What does cross-adsorbed mean?

The secondary has been passed over immobilised serum from other species to remove antibodies that would bind them. Use cross-adsorbed reagents for multiplexing and whenever species could collide on the blot.

HRP or fluorescent secondary?

Chemiluminescence with peroxidase is very sensitive and saturates easily, which makes quantitation awkward. Fluorescent secondaries give a wider linear range and easy two-colour multiplexing, and need an imager.

Why is my background so high?

Most often the secondary used at the dilution on the bottle. Titrate it on your own blot, and run a secondary-only control to see whether it binds anything directly.

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