Choosing developmental signalling antibodies by the pool you need to see: why a beta catenin antibody, and the beta-catenin antibody, b-catenin antibody and b catenin antibody spellings sold beside it, must separate the junctional pool from the signalling pool that only exists when the pathway is on, what a wnt3a antibody and a wnt5a antibody detect as secreted ligands and where an rnf43 antibody and an rspo3 antibody set receptor turnover, how a yap antibody and a lats2 antibody with a tead1 antibody report a pathway that also works by translocation, what an shh antibody and a gli2 antibody require of the cilium, why a dll1 antibody, a dll3 antibody, a dll4 antibody, a nicd antibody, a hes1 antibody, an atoh1 antibody and an ascl2 antibody read a pathway whose activation is a cleavage, and what a twist1 antibody, a zeb2 antibody, a snai1 antibody, an id3 antibody and a klf4 antibody or klf2 antibody report downstream

Choosing and controlling

1. Separate the structural pool from the signalling pool. For proteins that also sit at cell junctions, most of the signal in a lysate is structural. Reagents raised against the non phosphorylated signalling form, or fractionation and imaging, are what reveal the pool that matters.
2. Detect cleavage products, not receptors. Where a pathway activates by proteolysis, the intracellular fragment is the signal. An antibody against the extracellular region of the receptor will never report activation.
3. Treat translocation pathways as imaging problems. Factors that move between cytoplasm and nucleus need fractionation with validated markers, or per cell imaging. A whole cell blot averages the event away.
4. Measure secreted ligands where they act. Ligands in these pathways are lipid modified, poorly soluble and act locally. Lysate measurements are unreliable and conditioned medium is often the only informative sample.
5. Anchor with a pathway agonist and antagonist. A known activator and a known inhibitor bracket the assay. Given how much of the signal in these pathways is constitutive, that bracket is what makes a modest change believable.

Two pools, one antibody

The recurring difficulty in this area is that a single protein does structural work and signalling work, and a catalogue antibody sees both. That is why the reagents that matter are the ones raised against a modification or a conformation rather than against the protein.

Where such a reagent does not exist, change the readout rather than the antibody: a transcriptional reporter, a target gene panel or a subcellular measurement will answer the question the blot cannot.

Downstream targets as evidence

Because activation is hard to see directly, the induction of well established target genes is frequently the more robust readout. It is indirect and it is measurable, reproducible and quantitative.

Choose targets that are specific to the pathway in your cell type rather than the canonical ones from another system, and validate them once with an agonist and an antagonist.

Common questions

Why is my signal high in unstimulated cells?

Because most of the protein is in a structural pool at junctions or in a stable complex. Use a reagent specific to the active form, or a subcellular readout, rather than trying to interpret total lysate.

How do I detect activation of a cleavage pathway?

With a reagent raised against the neoepitope created by cleavage, and by looking in the nucleus rather than at the membrane. Total receptor tells you about expression only.

Can these ligands be measured in a lysate?

Poorly. They are secreted, lipid modified and act over short distances, so conditioned medium or a reporter assay is usually a better measurement than any blot.

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