Western blot secondary antibody: host, label and dilution

The rules, in order, for an anti mouse secondary antibody or an anti rabbit secondary antibody

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 such as an anti goat secondary antibody

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.

Endogenous immunoglobulin is the usual background

Tissue and serum samples already contain immunoglobulin, and a secondary raised against that species will bind it enthusiastically. The result is a strong, plausible band at the heavy or light chain position and a smear that looks like a loading problem.

The answers are a secondary adsorbed against the species present, a conformation-specific reagent that avoids denatured chains, or a detection format that does not use a whole anti-species secondary at all. A secondary-only lane on every blot is what tells you which problem you have.

Primary antibodies are validated for the application, not just the target

A clone's performance on a denatured blot predicts very little about its performance on fixed tissue or on live cells, and the reverse is also true. Product pages list applications, and the useful question is what evidence sits behind each listing rather than how many are listed.

Where a supplier shows data for one application and claims three, treat the other two as untested. Buying validation for the application you will use is the cheapest way to avoid spending a month discovering the difference.

A dbh antibody and a noradrenergic marker

Dopamine beta hydroxylase is in the vesicles of noradrenergic neurons and chromaffin cells, so a dbh antibody gives a granular pattern in processes rather than a filled cytoplasm and marks a population distinct from the dopaminergic one. It is also secreted, so plasma activity is a separate measurement, and the tissue's own expected pattern is the control.

A pin1 antibody and an isomerase that changes a substrate

PIN1 changes the conformation of phosphorylated substrates rather than modifying them, so a pin1 antibody reports the enzyme while the effect is read on a substrate's stability or location. Its own level is stable, so a knockdown or an inhibitor is what supports a functional claim, and the substrate chosen has to have a documented dependence.

An anti rabbit fluorescent secondary antibody and the linear range

An anti rabbit fluorescent secondary antibody gives a wider linear range and lower sensitivity than an enzymatic one, which is what makes it the choice for quantitative blots and multiplexing two channels on one membrane. Membrane autofluorescence and the imager's channels decide which dyes can be used, and the dye rather than the host is what has to match the instrument.

Common questions

How do I choose a secondary antibody western blot detection needs?

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 antibodies?

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.

Why do I see bands at the immunoglobulin chain positions?

The secondary is binding immunoglobulin already present in the sample. Use a secondary adsorbed against that species, or a conformation-specific reagent, and run a secondary-only lane to confirm the source.

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