What makes a target difficult and what the antibody has to prove: why a muc16 antibody must be validated against a heavily glycosylated repeat structure whose epitope may be masked, what a glypican 3 antibody, an nnmt antibody, an lyve1 antibody, an arl13b antibody and a fibrinogen antibody each demand of fixation, extraction and controls, where an anti-mcherry antibody against an introduced tag is the easiest validation available and why it is still worth doing, and how to decide whether a reagent has been validated for your application or merely sold for it

Choosing for a difficult target

1. Identify the obstacle before the reagent. Glycan masking, membrane anchoring, low abundance, high background from an abundant relative and localisation that fixation destroys are different problems. Naming yours narrows the reagent list immediately.
2. Read the immunogen against the structure. For repeat containing and heavily modified proteins, where the immunogen sits determines whether the epitope is accessible in your preparation. A datasheet naming only the protein is not enough.
3. Match extraction to where the target lives. Membrane anchored and matrix associated proteins need extraction conditions a standard lysis buffer does not provide. A target that appears absent is frequently a target left in the pellet.
4. Choose fixation for the epitope and the structure. Some structures survive only in particular fixation, and cross linking masks epitopes that then need retrieval. This is where most immunostaining of difficult targets fails and where a pilot on a positive control tissue pays for itself.
5. Use a tagged construct as the validation shortcut. Where the biology allows, expressing a tagged version and detecting the tag gives a clean positive control and calibrates what a real signal looks like. It is the cheapest validation available.
6. Demand reduction evidence. Knockout, knockdown or a tissue known to lack the target is what distinguishes a validated reagent from a sold one. Where the supplier has none, generate it yourself once and keep the image with the reagent record.

Validated for what, exactly

A reagent validated for denatured protein on a membrane may be useless on a fixed section, and one validated on an overexpressing cell line may not see endogenous levels. The application and the abundance are both part of the claim.

Read what was actually shown rather than which applications are listed. Listed applications are frequently aspirational; the images in the datasheet are evidence.

Two reagents beat one

Where a conclusion rests on the presence or distribution of a protein, two independent antibodies against different epitopes agreeing is far stronger than one antibody with a good datasheet. Disagreement between them is equally informative and worth knowing early.

It costs one extra reagent and one extra experiment, and it removes the most common criticism of this kind of result.

Common questions

Why does an antibody fail on a glycosylated target?

Because the glycan physically blocks the peptide epitope in the native protein, even though the immunogen worked. Reagents raised against accessible regions or against the glycan itself, and deglycosylation controls, are the routes around it.

Is a tag antibody a substitute for a target antibody?

Only for the tagged construct, and it says nothing about the endogenous protein. It is extremely useful as a positive control and as a calibration of what real signal looks like.

How much validation is enough?

Enough that someone else would believe the signal is the target: a reduction control, an appropriate positive and negative sample, and consistency between two independent reagents where the conclusion depends on it.

What if no validated reagent exists?

Say so in the write up and choose a design that does not depend on the antibody alone, for example a tagged construct, a genetic readout or mass spectrometry. An unvalidated reagent presented as validated is the worse option.

Browse by service class

• Compare Gene synthesis on advertised prices