PCNA antibody and other nuclear markers: what each reports about a cell's state

What each marker reports

1. A proliferation marker reports recent history, not a snapshot. The protein accumulates through the cell cycle and persists after the cell leaves it, so staining reports cells that have cycled recently as well as those cycling now. Where the question is which cells are in S phase at this moment, a nucleotide incorporation assay answers it and an accumulation marker does not.
2. Choose the proliferation marker to match the question. Different proliferation antigens have different persistence and different cell cycle profiles, so they give different indices on the same tissue. Report which one was used and do not compare an index measured with one against a published figure measured with another.
3. Handle cytoskeletal staining as a fixation problem. Microtubules depolymerise in the cold and are poorly preserved by some fixatives. Aldehyde fixation at temperature, or a methanol protocol, each preserve different aspects, and a disappointing cytoskeletal image is nearly always fixation rather than antibody.
4. Anchor every count to a nuclear stain. A proliferation index is a fraction, so the denominator has to be counted. A general nuclear marker counterstain in a separate channel is what makes the fraction measurable rather than estimated, and it also exposes tissue folds and debris.
5. Run the controls that make the index credible. A tissue known to be proliferating, one known not to be, and a no-primary control on the same run at the same settings. An index reported without them is an impression with a number attached.

Quantifying an index honestly

Fix the acquisition settings from a control section and do not change them across a cohort, define the region and the threshold before looking at the images, and count enough cells that the fraction has a meaningful confidence interval.

Auto-exposure across a cohort makes every section look equally stained, which is the opposite of the measurement. This is the most common way a proliferation index stops being a measurement.

Cytoskeletal imaging beyond the stain

Post-translational modifications of tubulin mark stable against dynamic microtubules and are detected by separate antibodies, which is a considerably more informative experiment than total tubulin staining.

For live dynamics, a fluorescent protein fusion or a small molecule probe answers questions fixation cannot. A fixed image of a dynamic structure is a single frame of a film.

Buying the reagent

Prefer a clone with published images in your tissue and your fixation, and prefer a recombinant monoclonal for a study running over years. For polyclonal reagents, buy one lot for the whole cohort and record it.

Titrate on your own sections rather than using the datasheet dilution. Proliferation markers in particular are frequently used far too concentrated, which raises background and inflates the index.

An aurora b antibody and the midzone it marks

Aurora B moves from the centromere in metaphase to the midzone in anaphase, so an aurora b antibody reports a position in mitosis rather than a level, and the pattern is what says the stain is real. Its activity is read as phosphorylation of histone H3 at serine 10, which is a different antibody and the usual companion. A mitotic arrest is the control that provides cells to look at.

A cyclin d1 antibody and the pathology reading of it

Cyclin D1 is scored in pathology as nuclear positivity because the translocation that drives it produces a strong nuclear signal, so a cyclin d1 antibody is chosen for a section as often as for a blot. In culture it is short lived and growth factor dependent, which makes serum starvation the negative control. Cytoplasmic staining is an artefact rather than a finding in most tissues.

A cdk7 antibody and a kinase with two jobs

CDK7 both activates other cell cycle kinases and phosphorylates the polymerase tail during transcription, so a cdk7 antibody is used in two literatures with different readouts. Protein level is stable, so the informative measurements are the phosphorylation of its substrates and the effect of a selective inhibitor. It sits in a complex, so an immunoprecipitation brings its partners with it.

An acetylated alpha tubulin antibody and the modification it reads

An acetylated alpha tubulin antibody detects a modification on a residue inside the microtubule lumen that marks stable, long lived microtubules, which is why it stains cilia and centrioles strongly. A total tubulin blot on the same membrane is the denominator, because the ratio rather than the signal is the claim. Cold or drug induced depolymerisation is the control that should abolish it.

A rhoa antibody and activity that no blot reports

RHOA protein level barely changes while its activity cycles between GTP and GDP bound states, so a rhoa antibody measures expression and a pulldown with an effector domain measures activity. Confusing the two is the commonest error in this literature. The protein is small and abundant, and prenylation means part of the pool is membrane associated, so the fractionation carries information too.

A cytokeratin antibody and the pan clone question

A cytokeratin antibody is either a pan clone recognising several family members, which is what an epithelial identity question wants, or specific to one keratin, which is what a subtype question needs. Cocktails are sold for the first purpose and should list their members. Because keratins are abundant and insoluble, the extraction rather than the antibody is what usually decides a blot.

A vcp antibody and a very abundant hexamer

VCP is an abundant hexameric ATPase whose level rarely changes, so a vcp antibody is used as a loading reference, for validating a knockdown, or to follow its recruitment to damaged organelles and protein aggregates. Because it is abundant, overloading is easy and the band saturates. Mutations in it cause disease, so the epitope position matters when a patient sample is the subject.

Common questions

What does a pcna antibody actually show?

A protein that accumulates during the cell cycle and persists for a period afterwards, so it marks recently and currently cycling cells. It is an accumulation marker, not a snapshot of cells in S phase.

Are proliferation markers interchangeable?

No. They differ in cell cycle profile and in how long they persist, so the index they give on the same tissue differs. Always state which marker produced an index, and do not compare across markers.

Why does my tubulin antibody give a poor image?

Fixation, almost always. Microtubules depolymerise in the cold and some fixatives preserve them badly. Test an aldehyde protocol at temperature against a methanol protocol on a known positive before changing antibody.

Do I need a nuclear marker antibody or counterstain?

For anything quantitative, yes. A proliferation index is a fraction and the denominator has to be counted, which needs a general nuclear marker in its own channel. It also reveals folds and debris that would otherwise be counted.

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