ALK antibody and receptor tyrosine kinase reagents for a target that may be a fusion

Choosing for the obstacle

1. Choose the domain the fusion retains. Where a rearrangement is the target, the reagent must bind a region present in the fusion protein. An antibody against the lost portion will be negative in exactly the samples of interest.
2. Extract membranes, do not boil them. Multipass membrane proteins aggregate when heated and stay in the pellet with a standard buffer. Use a membrane protocol and follow the supplier's advice on denaturation temperature.
3. Expect glycosylation to broaden the band. Heavily glycosylated receptors run as smears and at sizes well above the predicted mass. A deglycosylation control confirms the identity of an unexpected band.
4. Treat multipass receptors as the hardest class. Reagents against seven transmembrane receptors have a poor reputation for specificity. Where a conclusion depends on one, a knockout or knockdown control is not optional.
5. Separate expression from activation. Receptor level and receptor phosphorylation are different measurements needing different reagents. Report both when the claim is about signalling rather than about presence.

Membrane proteins fail before the antibody does

Poor solubilisation, aggregation on heating and conformational epitopes account for most negative results with receptor reagents. All three are protocol problems, and all three are usually diagnosed by blotting the pellet as well as the supernatant.

Do that once per cell type. It converts a recurring mystery into a known property of the sample.

Genetic controls settle the argument

For classes of target with a poor reagent record, a knockout or a strong knockdown showing loss of signal is the evidence that matters, and increasingly the evidence reviewers expect.

Where no genetic model is available, two independent reagents against different epitopes agreeing is the fallback, and it should be presented as such rather than as equivalent.

An fgfr1 antibody and the family it shares sequence with

The four fibroblast growth factor receptors are close relatives and the isoforms within each differ by a spliced extracellular loop, so an fgfr1 antibody has to name its epitope and its tested cross reactivity. Apparent mass moves with glycosylation, which is why the datasheet's own blot is the reference rather than the calculated weight. Pathway activation is read downstream through phosphorylated ERK and FRS2.

A ror1 antibody and a surface target with a narrow window

ROR1 is expressed in development and re-expressed in some cancers while staying low in adult tissue, which is what makes it a target and what makes a ror1 antibody hard to validate: most normal controls are negative by design. A positive cell line and a negative one from the same panel are the minimum, and for surface work the clone has to be validated on unpermeabilised cells.

A mertk antibody and the phagocytic reading

MERTK is read in macrophages and retinal pigment epithelium where it drives clearance of dying cells, so a mertk antibody is usually paired with an engulfment readout rather than used alone. The receptor is cleaved from the surface on activation, so a soluble fragment exists and an extracellular domain antibody may detect it. The family includes TYRO3 and AXL, which is the cross reactivity to check.

A pdgfr beta antibody and a pdgfrb antibody are one receptor

The receptor is catalogued under the spelled Greek letter and under the gene symbol, so a pdgfr beta antibody and a pdgfrb antibody can be the same clone in two listings, and searching one name hides part of the market. The alpha and beta receptors cross react easily, so the immunogen matters. It is read as a mural cell marker in tissue and as a phosphorylation in signalling work.

An erbb4 antibody and the isoforms cleavage produces

ERBB4 exists as juxtamembrane and cytoplasmic isoforms, and one of them is cleaved to release an intracellular fragment that enters the nucleus. An erbb4 antibody against the tail therefore reports both the full receptor and that fragment, which is the point in some experiments and a confound in others. The expected bands and the isoform the clone sees belong in the method before the blot is read.

An ephb2 antibody and the gradient it is read as

Eph receptors are interesting as gradients across a tissue rather than as a level in a lysate, so an ephb2 antibody is an imaging reagent whose readout is a spatial pattern. The family is large and conserved in the kinase domain, so a clone raised there reports several members. Ligand binding triggers internalisation, which means surface staining falls in exactly the conditions the experiment is testing.

Common questions

Why is my RET antibody receptor blot at the wrong size?

Glycosylation, usually, which raises apparent mass and broadens the band. A deglycosylation treatment collapsing the band to the predicted size confirms the identity.

Why are GPCR antibodies distrusted?

Because many published reagents have failed knockout validation. It is a well documented problem in the field and it means a genetic control is required for any claim resting on one.

How do I detect a fusion protein?

With a reagent against a region retained in the fusion, ideally combined with an orthogonal method such as sequencing or a break apart assay. The domain choice is the whole question.

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