Choosing receptor kinase antibodies against a target that may be a fusion: why an alk antibody used to detect a rearrangement has to bind a region retained in the fusion protein and why the wild type receptor is silent in most tissues, what a ret antibody, a pdgfra antibody, a pdgfr beta antibody, a pdgfrb antibody, an fgfr1 antibody, an fgfr antibody, a ddr1 antibody, a ddr2 antibody, an ephb2 antibody, a kdr antibody, a vegfr2 antibody, a vegfr3 antibody, a neuropilin-1 antibody and a plexin d1 antibody each demand of extraction and phosphorylation state, how an asgr1 antibody, an lrp1 antibody, an npc1 antibody, an sglt1 antibody, an sglt2 antibody, a slc26a6 antibody, a cftr antibody and an atp1a1 antibody or na k atpase antibody behave as multipass or heavily glycosylated membrane proteins, and where a gpcr antibody, a glp-1r antibody, a glp1 antibody, a cb1 antibody, an s1pr1 antibody, a ptger2 antibody and an ffar2 antibody sit as the hardest class of all

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.

Common questions

Why is my 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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