An organelle marker is used to say where something is, so the reagent is judged on the pattern it gives rather than on a band's size, and the fixation decides whether that pattern appears at all. Every marker in this class also moves: the Golgi fragments in mitosis and under stress, cilia assemble and disassemble, granules form only when the cell is stressed.
An arl13b antibody and a syntaxin 6 antibody on cilia and the trans-Golgi
The small GTPase is the standard primary cilium marker, and its usefulness rests on the axoneme's own geometry: a short rod per cell, which makes a count and a length measurement possible and a diffuse stain obviously wrong. Serum starvation is the usual control, since ciliation follows the cell cycle. The trans-Golgi SNARE marks a compartment that is fragmented by brefeldin A and by mitosis, which is what makes it a useful readout of trafficking and a poor loading control.
An mtco2 antibody and a lamin a antibody on mitochondria and the envelope
A mitochondrially encoded subunit is a compartment marker and a genome readout at once: its abundance depends on mitochondrial DNA and translation, so it falls in models of mitochondrial disease rather than staying put as a control. Lamin A is the nuclear envelope marker used to show a nuclear fraction is really nuclear, and it is also a disease protein whose processing matters, so a clone's epitope decides whether prelamin A or the mature form is seen.
An arf1 antibody and a tia1 antibody on trafficking and stress granules
The small GTPase cycles between membranes and cytosol with its nucleotide state, so a fractionation rather than a total blot is what reports its behaviour, and a brefeldin treatment is the positive control. The granule protein is diffuse in an unstressed cell and forms puncta under arsenite or heat, which means the informative image is a treated sample beside an untreated one, and a diffuse stain is the negative control rather than a failure.
A beta tubulin antibody and an ago2 antibody, a control and a body marker
Tubulin is used as a loading control and as a cytoskeletal marker, and it is abundant enough to saturate a blot long before a scarce target is in range, so it belongs in the same exposure or it belongs nowhere. Argonaute 2 marks the silencing machinery and concentrates in processing bodies under stress, which is again a treated-against-untreated image rather than an abundance measurement. Neither is a loading control in a treatment that changes the cytoskeleton or the stress response.
Fixation decides the pattern
Methanol preserves microtubules and many peripheral scaffolds and destroys some membrane epitopes; paraformaldehyde preserves membranes and needs permeabilisation, whose detergent then decides which soluble proteins wash out. Golgi and granule patterns are especially sensitive, so a clone validated under one fixation is not validated under the other. The practical route is to fix the protocol on control cells, record it, and choose among clones documented under it.
Colocalisation, and what it can support
Two markers overlapping in a widefield image support very little, because the depth of field includes structures above and below. A colocalisation claim needs optical sectioning, channels checked for bleed-through with single stains, and a quantitative measure with its own control, either a known colocalising pair or a deliberately non-colocalising one. Reporting the measure and the instrument is what makes the claim survive review, and a merged image alone does not.
Markers that move, and how to handle it
Every marker here changes with the cell's state, which is a feature when the change is the readout and a trap when the marker is being used as a reference. The rule that follows is simple: a compartment marker is a reference only in an experiment that does not perturb that compartment, and where a treatment might, a second independent marker for the same compartment is added. That is cheaper than discovering after the fact that the denominator moved with the numerator.
What to ask for on this class
Ask for an image rather than a blot: the vendor's own staining figure in a cell type like yours, with the fixation and permeabilisation stated, and ideally a knockdown or knockout image beside it. Then ask about the counterstain and the channel, since a marker's usefulness depends on being free in the panel you already run. For anything quantitative, ask whether the clone has been used for the measurement you intend, because pattern and quantification are different demands.
Questions people ask about giantin antibody
Can beta-tubulin be a loading control?
For gross loading only, in the same exposure as the target, and never in an experiment that changes the cytoskeleton.
Why does the Golgi look fragmented?
It fragments in mitosis and under stress and drug treatment. That is a readout rather than a staining failure, which is why the marker is a poor reference.
What does a colocalisation claim need?
Optical sectioning, bleed-through controls, a quantitative measure and a control pair. A merged widefield image is not evidence.