Beta catenin antibody and developmental pathway reagents: choose by the pool you need to see

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.

A creb antibody and a factor read by its phosphorylation

CREB protein level is constant while its activity comes from phosphorylation at a named serine, so a creb antibody is the denominator and a phospho clone is the measurement, with both on the same membrane. The phosphorylation is fast and transient, so a short time course is the experiment. A family relative shares the epitope region, which is what cross reactivity data are for.

A wt1 antibody and a marker with two readings

WT1 is read as a lineage and tumour marker in pathology and as a transcription factor in kidney development, so a wt1 antibody is interpreted against the tissue and against the clone's validated application. Nuclear staining is the expected pattern and a cytoplasmic signal is usually the dilution or the retrieval. Several isoforms arise from alternative splicing and start sites.

An ap2 antibody and the two things that name means

An ap2 antibody may target the AP-2 transcription factor family or the AP-2 clathrin adaptor complex, which are unrelated proteins with the same abbreviation, so the catalogue entry has to be read for the gene rather than the name. For the adaptor, the subunit is the identity and the readout is recruitment to a membrane; for the factor it is nuclear staining.

An srebp2 antibody and a factor cleaved to act

SREBP2 is held in the endoplasmic reticulum membrane and is cleaved when sterols fall, so the active species is a smaller nuclear fragment and an srebp2 antibody has to say which form its epitope sees. Sterol depletion with a statin and a lipid free medium is the control that produces the fragment. The full length and cleaved bands both belong in the figure.

A pka antibody and a holoenzyme rather than a protein

Protein kinase A is a complex of catalytic and regulatory subunits, so a pka antibody names a subunit and activity is read as the phosphorylation of a substrate motif rather than as a level. A phospho-substrate antibody against that motif is the usual companion reagent. Because the subunits dissociate on activation, a native preparation can show the complex and a denatured one cannot.

An lck antibody and a kinase read in T cells

LCK sits at the top of the T cell receptor cascade and is regulated by two phosphorylation sites with opposite effects, so an lck antibody for total protein is read with phospho clones that name their residue. It is myristoylated and partly membrane associated, which makes the fraction informative. A T cell line is the positive control, since expression is restricted.

An hdac2 antibody and the paralogue it cannot be told from

HDAC1 and HDAC2 are close paralogues in the same complexes, so an hdac2 antibody needs cross reactivity data and a single knockdown often produces no phenotype because the other compensates. Activity belongs to an enzyme assay. Complex membership is read by immunoprecipitation with the corepressor subunits, which is the experiment that makes an HDAC finding mean something.

A bcat1 antibody, which is not a beta catenin antibody

A bcat1 antibody reports branched chain aminotransferase 1, a metabolic enzyme, and not beta catenin, whose gene is CTNNB1: the similar abbreviation is a genuine ordering trap and the two have nothing to do with each other. Checking the gene identifier rather than the abbreviation is the habit that prevents it.

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, for example with an NICD antibody?

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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