RB1 antibody and other tumour suppressor markers: proving presence, and proving loss

Reading each one correctly

1. Read RB1 as a phosphorylation state, not an amount. The protein is active when hypophosphorylated and inactivated by phosphorylation through the cycle, so a total antibody reports presence while a phospho-specific reagent reports the state that matters. Say which you used and at which residue, because the two support different conclusions.
2. Treat loss of expression as the hardest claim to make. Showing a protein is absent requires an antibody that would certainly have detected it, which means a positive control on the same slide or membrane and, ideally, a graded series. An absent band on a blot with no positive lane demonstrates nothing about the sample.
3. Count DNA damage markers as foci, not as intensity. Damage response proteins concentrate into discrete nuclear foci at break sites, so the readout is foci per nucleus over time. Total nuclear intensity averages the signal away and is the commonest way a damage experiment reports nothing.
4. Include an inducer and a time course. A known damaging treatment produces foci that appear and then resolve, which demonstrates the assay works and gives the kinetics that are usually the interesting result. Without an inducer, an absence of foci is uninterpretable.
5. Use an isogenic null where the biology allows. For loss-of-function claims, a line known to lack the protein run alongside is the strongest available control, and for these genes such lines are widely available. It settles in one lane what an isotype control cannot.

Fixation and retrieval for nuclear targets

These are nuclear proteins and most need heat induced retrieval on fixed tissue. Test citrate near pH six against a Tris buffer near pH nine on a known positive, because the right condition differs between them and inheriting a protocol wastes weeks.

For foci counting, section thickness and imaging plane matter: a focus split across the top of a section is counted differently from one in the middle. Fix the acquisition and the counting rule before the cohort.

Quantifying without overreaching

Scoring loss in tissue as present or absent needs a defined threshold set on control material and applied blind. Thresholds chosen after seeing the cohort are the commonest reason a marker association does not replicate.

Where possible, include an internal positive control within the same section: stroma or infiltrating cells that should retain the protein make an absent tumour signal far more convincing than an adjacent control slide.

A kras antibody, and why pan and mutant specific differ

A kras antibody raised against the shared sequence reports the protein whatever its codon 12 state, which is the opposite of what a mutation specific clone does, so the two answer different questions and one cannot substitute for the other. The protein is small and abundant, prenylated and partly membrane associated. A molecular assay remains the reference for mutation status.

An arid1a antibody and loss as the finding

ARID1A is frequently mutated with loss of protein, so an arid1a antibody is used to show absence, and absence needs an internal positive population in the same section or a positive control on the same blot. The protein is very large and chromatin associated, which sets the extraction and the transfer. A patient derived line with known loss is the cleanest control available.

An fbw7 antibody and a ligase that sets other proteins' levels

FBW7 marks several oncoproteins for degradation, so an fbw7 antibody reports the ligase while the readout is a substrate's level, and that is why a figure resting on the ligase alone says little. It is catalogued under more than one symbol, which splits search results. Its own level is fairly stable, so a change in a substrate with no change in the ligase is the expected pattern.

An id3 antibody and a factor that works by binding others

ID3 has no DNA binding domain and acts by sequestering the factors that do, so an id3 antibody measures a small nuclear protein whose consequence is read on its partners' targets. The family is small and similar, so cross reactivity against ID1 and ID2 belongs in the datasheet. Expression is developmentally restricted, which makes the tissue control the interpretation's anchor.

A nur77 antibody and an immediate early nuclear receptor

NUR77 is induced within minutes of stimulation and then degraded, so a nur77 antibody reports a moment and a resting sample shows almost nothing, which is the expected result rather than a failure. It also moves between nucleus and mitochondria, so location is part of the finding. A short stimulation time course is the only way to see it.

An erg antibody and the fusion that drives it

ERG is overexpressed in prostate cancer through a gene fusion, so an erg antibody is scored as nuclear positivity that indicates the rearrangement, and the vascular endothelium in the same section is positive by nature, which serves as an internal control. The family shares a DNA binding domain, so the clone's specificity against related factors decides whether the call is safe.

Common questions

Total or phospho-specific for RB1?

Phospho-specific if the question is about the protein's activity, because it is inactivated by phosphorylation rather than by degradation. A total antibody answers whether the protein is present, which is a different and usually easier question.

How do I show a tumour suppressor is lost, for example with a CDKN2A antibody?

With a positive control that would certainly have been detected, on the same membrane or slide, and preferably an isogenic null as a negative. Absence of signal in isolation is not evidence of absence of protein.

Why does my 53BP1 antibody stain look like nothing happened?

Almost always because the readout was total nuclear intensity rather than foci. These proteins concentrate at break sites, so count discrete foci per nucleus and run a time course after a known inducer.

Does a transcript measurement substitute?

Not for these. Loss frequently happens by deletion, by promoter methylation or by protein destabilisation, and transcript and protein disagree often enough that the protein measurement is the one that supports the claim.

Browse by service class

• Compare Gene synthesis on advertised prices