Detecting nuclear proteins that are scarce, structural or transient: why a telomerase antibody targets an enzyme present at very low copy number and why activity assays usually beat detection for it, what a trf2 antibody reports about telomere protection, how a lamin a antibody, a lamin a/c antibody and a lamin antibody or lamin b antibody differ in the isoform they see and why that matters for nuclear envelope work, where a tia1 antibody and a tia-1 antibody mark stress granules rather than nuclei, what an ago2 antibody, an upf1 antibody, a gspt1 antibody, an hnrnp a1 antibody, an hnrnp k antibody, a ptbp1 antibody and a yb1 antibody demand as RNA binding proteins that move between compartments, and why a brdu antibody, brdu antibodies generally and an anti-brdu antibody still need a denaturation step nothing else on this page requires

Getting nuclear stains and blots to work

1. Extract the nucleus rather than the cell. Chromatin and envelope associated proteins stay in the pellet with a standard buffer. A nuclear extraction, with the pellet checked, is what puts them on a blot.
2. Prefer activity where abundance is very low. For enzymes present at a handful of copies per cell, an activity assay is more sensitive and more meaningful than an attempt to detect the protein directly.
3. Name the isoform for envelope proteins. Splice isoforms of nuclear envelope proteins differ in tissue distribution and in disease relevance, and reagents differ in which they see. The datasheet has to state it.
4. Treat RNA binding proteins as mobile. These proteins relocalise into granules and between compartments on stress. A change in staining pattern is frequently relocalisation rather than expression, and fractionation or imaging is what distinguishes them.
5. Denature before detecting incorporated nucleotide. An antibody against an incorporated base cannot reach it in double stranded DNA. Acid or enzymatic denaturation is a required step, and it damages other epitopes in the same sample.

Compartment before reagent

For this group, where the protein is matters more than which antibody is used. A reagent that works beautifully on a nuclear extract will show nothing in a cytoplasmic lysate, and the reverse is equally true.

Fractionate once with validated markers and the question of where each target lives in your cells is settled for good.

Relocalisation looks like regulation

Proteins that move between compartments produce apparent changes in expression when only one compartment is examined. That is a systematic error rather than noise, and it points in a consistent direction.

Where a target is known to move, image it or fractionate. A whole cell blot cannot see the event that matters.

Common questions

Why can I not detect telomerase?

Because it is present at extremely low copy number in most cells. Activity assays are the standard approach, and a detection attempt without enrichment is likely to fail whatever the reagent.

Does a stress granule marker measure stress?

It measures granule formation, which is one response to some stresses. Total protein does not change, so the measurement is the number and size of granules per cell, which is an imaging readout.

Why does incorporated nucleotide detection need denaturation?

The epitope is inside double stranded DNA and inaccessible. Denaturation exposes it and damages protein epitopes at the same time, which is why co-staining needs a compatible protocol or a different label chemistry.

Browse by service class

• Compare Gene synthesis on advertised prices