Biochemistry · Separations Technique

The Centrifuge Bench

Four ways a spinning rotor sorts molecules by how fast — or how far — they sediment through a liquid column. Press run on any tube to watch the separation play out.

large / dense particle (e.g. a nucleus, a large complex)
medium particle (e.g. an organelle, a ribosome)
small particle (e.g. a free protein)
density gradient, pale (low) → deep (high)
01 — Differential centrifugation

Sequential spins, sorted by size

crude fractionation of a cell homogenate

A homogenate is spun at low speed to pellet the largest, heaviest structures. The supernatant is poured off and re-spun at higher speed to pellet the next size class, and so on — each round clearing out whatever is left.

1,000 × g → nuclei 20,000 × g → mitochondria 100,000 × g → ribosomes
02 — Rate-zonal centrifugation

A thin layer, raced through a gradient

sedimentation velocity

Sample is loaded as a narrow band on top of a pre-formed sucrose gradient, then spun for a timed interval — stopped well before equilibrium. Particles sediment at rates set by their size and shape, so they resolve into separate bands part-way down the tube.

timed run, not to equilibrium separates by size & shape
03 — Isopycnic (equilibrium) centrifugation

Settling to a matching density

buoyant-density / equilibrium sedimentation

Sample is mixed throughout a steep gradient (classically CsCl) and spun until equilibrium. Each particle migrates — up or down — until the surrounding solution matches its own buoyant density, then stops moving no matter how much longer the run continues. Separation depends on density alone, not size.

run to equilibrium separates by density only e.g. Meselson–Stahl
04 — Analytical ultracentrifugation

Watching the boundary move

real-time optical detection

Nothing is isolated here — a spinning sample cell is scanned optically while it spins, tracking the sedimenting boundary as it moves outward from the center over time. The boundary’s speed gives the sedimentation coefficient; equilibrium runs give molecular weight, purity, and shape.

scans at successive times gives S & Mr, not a pellet