Subcellular fractionation
A series of spins of rising force, each collecting whatever sediments at that force and passing the rest on. Nuclei come down at a few hundred times gravity, mitochondria at ten thousand, microsomes only in an ultracentrifuge, and the cytosol stays in the final supernatant. Each pellet is enriched rather than pure.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Differential centrifugation
A series of spins of rising force, each collecting whatever sediments at that force and passing the rest on. Nuclei come down at a few hundred times gravity, mitochondria at ten thousand, microsomes only in an ultracentrifuge, and the cytosol stays in the final supernatant. Each pellet is enriched rather than pure.
How it differs from the standard method
- Forces and times transfer between rotors only as × g and minutes, never as rpm.
- The first spin is the one to be careful with: above about 1,000 × g the nuclear pellet starts collecting mitochondria.
- Washing each pellet once at the same force roughly halves the carry-over from the supernatant it was spun out of.
- Mitochondria, lysosomes and peroxisomes sediment together, so this method alone cannot separate them.
- Yield and purity move in opposite directions at every spin, and both have to be reported for the result to mean anything.
1Separating by how fast things sink
A cell broken open gently is a suspension of intact organelles, and they sediment at very different rates: nuclei in seconds at a few hundred times gravity, mitochondria in a quarter of an hour at ten thousand, ER vesicles only in an ultracentrifuge, and soluble cytosolic protein essentially not at all. Differential centrifugation exploits exactly that.
2Every pellet is enriched, not pure
This is the single most important thing to understand about the method, and the thing most figures get wrong. Sedimentation rate is a continuum, not a set of bins.
Nuclear pellet
Nuclei and unbroken cells. Go above about 1,000 × g and mitochondria start coming down with them.
Mitochondrial pellet
Mitochondria, with lysosomes and peroxisomes, which sediment at similar rates. Only a gradient separates those.
Microsomal pellet
ER and Golgi vesicles, plasma-membrane fragments. Needs an ultracentrifuge because the vesicles are small.
Cytosol
Soluble protein at a few svedbergs, which no practical spin pellets. Its purity depends on how well the earlier spins worked.
3Homogenisation is the step that decides everything downstream
The aim is to open the plasma membrane and nothing else. Cells are swollen first in a hypotonic buffer, which puts the plasma membrane under tension so that a few strokes of a Dounce pestle shear it open, and the medium then contains 0.25 M sucrose or mannitol to keep the organelles at the osmotic pressure they are used to.
4Density gradients buy purity with time and yield
Differential centrifugation cannot separate two organelles that sediment at the same rate, and mitochondria, lysosomes and peroxisomes do. A density gradient separates by buoyant density instead: the sample is layered on a gradient of Percoll, sucrose, iodixanol or Ficoll, spun until each particle either reaches its own density (isopycnic) or has travelled a characteristic distance (rate-zonal), and the gradient is then collected fraction by fraction.
5Nuclear extraction as a two-step special case
The commonest fractionation in practice is the simplest: separate cytoplasm from nucleus, then pull the nuclear proteins out with salt. Cells are swollen in hypotonic buffer, often with a trace of a non-ionic detergent, and broken with a few strokes or by vortexing; the nuclei are pelleted at a few hundred times gravity, washed, and extracted with 300–420 mM NaCl, which is what releases transcription factors from chromatin.
Key terms
Practise these as flashcards →The share of a marker found in a fraction divided by the share of the total protein in that fraction. It is the number a marker blot actually supports, and 1.0 means the fraction is no better than the homogenate.
A series of spins of increasing force, each collecting the class of particle that sediments at that force while leaving the slower ones in the supernatant.
Separation by sedimentation rate down a gradient, stopped before anything reaches equilibrium. Position depends on size as well as density, so the run time matters.
Separation run to equilibrium, so each particle stops where the medium matches its own buoyant density and position no longer depends on run time.
What is left after the first low-speed spin: everything except nuclei and unbroken cells. The starting material for the mitochondrial spin.
A protein known to be restricted to one organelle, blotted to show where that organelle ended up. The contaminating markers are the controls.
Sucrose or mannitol in the homogenisation medium at a concentration that keeps organelles from swelling and bursting while the cell is being broken.
A glass mortar and a close-fitting glass pestle. The clearance sets the shear, so the same number of strokes means different things in different pestles.
Contamination from the film of supernatant a pellet is wet with, which a single wash at the same force largely removes.
When to use it
- Asking where in the cell a protein is, or whether it moves between compartments after a treatment, when an antibody good enough for imaging is not available.
- Preparing enriched organelles as starting material: mitochondria for a respiration or import assay, microsomes for a translocation assay, nuclei for a chromatin method.
- Separating cytoplasmic from nuclear protein to show that a transcription factor has translocated, which is the commonest use by a wide margin.
- Reducing the complexity of a proteomic sample, where enriching a compartment several fold brings its low-abundance proteins into range.
Compared with alternatives
The underlying method; go there for how force, rotor geometry and run time combine, and for gradient mechanics in general.
Prefer it when the question is localisation in single cells and a good antibody exists; it sees heterogeneity that a fraction averages away.
The alternative when total protein is what you need; a whole-cell lysate answers abundance questions that fractionation only complicates.
The readout that makes a fractionation interpretable, since enrichment and cross-contamination are both measured on the marker panel.
Often the destination: enriched fractions plus quantitative proteomics is how organelle inventories are built.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A series of spins of rising force, each collecting whatever sediments at that force and passing the rest on. Nuclei come down at a few hundred times gravity, mitochondria at ten thousand, microsomes only in an ultracentrifuge, and the cytosol stays in the final supernatant. Each pellet is enriched rather than pure.
- Forces and times transfer between rotors only as × g and minutes, never as rpm.
- The first spin is the one to be careful with: above about 1,000 × g the nuclear pellet starts collecting mitochondria.
- Washing each pellet once at the same force roughly halves the carry-over from the supernatant it was spun out of.
- Mitochondria, lysosomes and peroxisomes sediment together, so this method alone cannot separate them.
- Yield and purity move in opposite directions at every spin, and both have to be reported for the result to mean anything.