CD81 antibody and extracellular vesicle markers: marking without over-claiming

Characterising a preparation

1. Use at least three positive markers. Single marker characterisation is not accepted, because each marker is present on a subset and on other material. A panel spanning different marker classes describes the preparation far better than any one of them.
2. Include a negative marker. An abundant intracellular protein from a compartment that should not be in the preparation shows whether cell debris or lysed material contaminates it. This is the control that most preparations lack and most reviewers ask for.
3. Report the isolation method with every result. Differential centrifugation, density gradient, size exclusion and precipitation yield different populations with different contaminants. Two preparations by different methods are not comparable, and the method belongs in the figure legend.
4. Measure particle number and protein separately. Protein per particle differs enormously between preparations and between methods. Reporting only protein makes a contaminated preparation look abundant.
5. Do not infer origin from markers alone. Marker enrichment supports a claim about vesicle type; it does not establish biogenesis route. Where origin matters, genetic perturbation of the pathway is what supports it.

Enrichment is the honest word

The vocabulary in this field runs ahead of the evidence, and marker panels are frequently presented as identifying a biogenesis route they cannot establish. Writing enrichment where enrichment is what was shown costs nothing and makes the work durable.

Community guidance in this area has converged on multiple positive markers, at least one negative marker and full reporting of the isolation method. Following it removes the most common criticism of these papers.

The preparation is the experiment

More variance in vesicle work comes from isolation than from detection. Serum in the culture medium contributes its own particles, cell viability changes what is released, and centrifugation conditions select different populations.

Use depleted medium, record viability, fix one method and report it. Those four steps account for most of the reproducibility difference between laboratories.

An xct antibody and the transporter cystine comes in through

The cystine glutamate exchanger is SLC7A11, read in ferroptosis work, and an xct antibody carries the same handling problems as any multipass transporter: gentle lysis, no boiling, a membrane preparation rather than a whole cell lysate, and a broad band. It works as a heterodimer with a partner chain, so the partner belongs on the same blot when the complex is the claim.

A glut2 antibody and the family it shares folds with

The glucose transporters are a family of multipass proteins with shared topology, so a glut2 antibody needs cross reactivity data against the relatives expressed in the same tissue. Expression is restricted to liver, pancreas and intestine, which is what makes a tissue positive control meaningful. Apparent mass sits below the calculated value because these proteins run anomalously on a gel.

An slc26a6 antibody and an anion exchanger read by location

Transport function depends on which membrane a protein reaches, so an slc26a6 antibody is read as apical or basolateral staining in a polarised epithelium rather than as a level, and a polarity marker in the same image is part of the measurement. Several splice forms exist, so the expected mass depends on the isoform, and a knockout tissue is the validation worth looking for.

An asct2 antibody and glutamine uptake

ASCT2 is SLC1A5, the main glutamine transporter in many tumour lines, so an asct2 antibody is read beside an uptake measurement rather than instead of one. It is glycosylated and multipass, giving a broad band, and it is often upregulated rather than switched on, which makes a quantitative comparison with a stated loading reference the only useful readout.

An sglt1 antibody and the sodium coupled route

SGLT1 sits apically in intestine and kidney and is the target of a drug class aimed mostly at its relative, so an sglt1 antibody must be specific against SGLT2 and the datasheet has to say so. As with every transporter here, boiling aggregates it and a whole cell lysate dilutes it, so a membrane fraction and a stated sample preparation are what produce a band.

An slc3a2 antibody and the chaperone chain of the complex

SLC3A2, also called CD98 heavy chain, pairs with several light chains including the cystine and the amino acid transporters, so an slc3a2 antibody reports the shared partner rather than one transport activity. That makes it useful for showing a complex is assembled and unhelpful for naming which transporter is present. The two chains are disulphide linked, so reducing conditions change the band.

Common questions

Is an exosome antibody stain, such as a CD63 antibody, proof of exosomes?

No. Those markers are enriched on small vesicles and are also found on the plasma membrane and other particles. They support a claim of enrichment, and origin needs perturbation of the biogenesis pathway.

Which negative marker should be used?

An abundant protein from a compartment that should be absent, run on the same blot with equal loading. Its absence is what distinguishes a vesicle preparation from a preparation of cell fragments.

Why do preparations differ so much between laboratories?

Because isolation method, starting material and culture conditions all change what is recovered. Reporting the method in full is the minimum requirement for anyone else to interpret the result.

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