TOM20 antibody and mitochondrial markers that report the right thing

Choosing mitochondrial reagents

1. Separate mass from function first. If the question is how much mitochondria a cell has, use a marker known to scale with mass. If the question is respiratory capacity, use subunits and enzyme activities and do not normalise them to each other.
2. Remember the two genomes. Respiratory complexes contain subunits encoded in the nucleus and in the mitochondrion, and they respond differently to stress and to drugs. A cocktail covering both is far more informative than any single subunit.
3. Match the reagent to the membrane. Outer membrane, inner membrane, intermembrane space and matrix proteins behave differently in fractionation and in permeabilisation experiments. Naming the sublocation is part of choosing the reagent.
4. Read shape proteins by imaging. Fusion and fission machinery levels say little about network morphology. The measurement that reports shape is imaging with a segmentation analysis, and the blot is at best supporting evidence.
5. Normalise carefully. Normalising a mitochondrial protein to a cytosolic loading control confounds mitochondrial content with expression. Where content is the variable, report both the mitochondrial marker and the total protein and let the reader see the ratio.

Mass and function pull apart

Cells adjust mitochondrial content and mitochondrial composition independently. A treatment can raise respiratory subunit levels without changing mass, or expand the network while diluting the chain. Measuring one and reporting the other is the standard confusion.

The honest presentation shows both: a mass marker and a functional readout, with the ratio stated rather than assumed.

Fractionation needs its own controls

A mitochondrial fraction contaminated with endoplasmic reticulum is common, because the two membranes are physically associated. Any fractionation claim needs markers for the contaminating compartments blotted alongside.

Show those controls in the figure. Without them a fractionation result is an assertion rather than evidence.

A drp1 antibody and the fission it is read for

Mitochondrial fission is measured as network morphology, so a drp1 antibody is read beside an outer membrane marker and the readout is the shape of the network rather than a band. Activity is set by phosphorylation at two residues with opposite effects, so a phospho clone naming the residue is the measurement and total protein is the denominator. Puncta on tubules are the expected pattern.

An mff antibody and the receptor that recruits the machinery

Fission needs receptors on the outer membrane to bring the cytosolic machinery in, and an mff antibody detects one of them, so it belongs in the same figure as the fission enzyme rather than alone. It is a small tail anchored protein with several isoforms, so the expected bands belong in the method. A membrane fraction rather than a whole lysate is where it is cleanly seen.

A vdac2 antibody and a vdac1 antibody across the porins

The outer membrane porins are close paralogues, so a vdac2 antibody and a vdac1 antibody both need cross reactivity data, and a pan reactive clone is useful only as a loading reference for the mitochondrial fraction. They run near thirty kilodaltons and are abundant, which makes them convenient normalisers. Which paralogue a figure means should be stated, because the two have different phenotypes.

A tfam antibody and the genome it packages

TFAM coats mitochondrial DNA, so a tfam antibody is read as nucleoid puncta inside the network and as a level that tracks copy number rather than as a diffuse signal. It is small and basic, so transfer is fast and overexposure is easy. An antibody against the DNA itself or a copy number measurement is what supports the claim the protein level suggests.

A citrate synthase antibody as the matrix reference

Citrate synthase is a stable matrix enzyme and its level is the conventional proxy for mitochondrial content, which is why a citrate synthase antibody is often run as the normaliser rather than as the subject. That makes it the control against which a change in another mitochondrial protein is read. Where content itself is the question, an activity assay is the usual companion measurement.

A prohibitin antibody and the inner membrane complex

Prohibitins form a ring complex in the inner membrane and are read for cristae organisation and for the complex rather than as single proteins, so a prohibitin antibody is often used with a native gel or a crosslinker. The two family members associate and destabilise without each other, so knocking one down lowers both, which is the result to expect rather than a failed experiment.

Common questions

Which marker is best for mitochondrial mass?

An abundant, stable structural protein rather than a respiratory subunit, because subunit levels respond to metabolic state. Confirm in your own system that the chosen marker tracks an independent measure of mass before relying on it.

Why measure subunits from both genomes?

Because nuclear and mitochondrially encoded subunits are regulated separately, and a mismatch between them is itself an informative finding about mitochondrial translation or genome maintenance.

Can an OPA1 antibody blot report mitochondrial morphology?

No. Fusion and fission protein levels are only loosely related to network shape, which has to be imaged and segmented. Reporting morphology from a blot is a claim the data does not support.

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