Centrifugation
A fixed-angle benchtop rotor taking 1.5–2 mL tubes, typically reaching 15,000–21,000 × g. The everyday workhorse for clearing lysates and digests, collecting alcohol precipitates, washing small pellets and spinning columns. Short path length means pellets form in minutes, but the modest force means anything slower than a few thousand svedbergs stays in the supernatant.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Microcentrifuge
A fixed-angle benchtop rotor taking 1.5–2 mL tubes, typically reaching 15,000–21,000 × g. The everyday workhorse for clearing lysates and digests, collecting alcohol precipitates, washing small pellets and spinning columns. Short path length means pellets form in minutes, but the modest force means anything slower than a few thousand svedbergs stays in the supernatant.
How it differs from the standard method
- Fixed-angle: the pellet forms on the tube wall at an angle, so take the supernatant off from the opposite side.
- Radii differ widely between models, so the same rpm is a different force: check the rotor's rmax before copying a speed.
- Refrigerated models need pre-cooling: the first minutes of an unprecooled run happen at room temperature.
1A field you can set, and a force you have to calculate
A centrifuge spins a rotor so that everything in the tube experiences an outward field far stronger than gravity. Particles denser than the surrounding liquid move outward through it, down the tube, at a speed set by their size, their shape, the difference between their density and the liquid's, and the viscosity of that liquid.
Force
Radius in cm, speed in rpm. Doubling the speed quadruples the force; doubling the radius only doubles it.
Time
A rotor's clearing factor k divided by a species' sedimentation coefficient gives the hours needed to drive it from the meniscus to the bottom.
Temperature
Everything sediments about a third more slowly in the cold room. Worth paying for a lysate; not free.
2Differential pelleting: one sample, several spins
Because sedimentation rate spans orders of magnitude across the contents of a cell, a series of spins at increasing force separates them. Whole cells and nuclei come down at a few hundred to a thousand × g in minutes.
3Gradients: rate-zonal and isopycnic
Pelleting is a blunt instrument: anything sedimenting faster than your cut-off comes down with your target. A density gradient gives a second dimension.
4How it goes wrong
- Copying rpm instead of × g from a paper written with a different rotor: the most common quiet error in the whole technique.
- Running too short: the supernatant is still turbid and the carryover appears as background in everything downstream.
- Running too long on a gradient: the heavy species pass the bottom and become a pellet that cannot be fractionated.
- Braking a gradient. Sudden deceleration swirls the liquid and remixes bands that took hours to form.
- An unbalanced rotor. At benchtop speeds it vibrates and wears the drive; in a high-speed rotor an imbalance can wreck the rotor and the instrument.
- Tubes that do not match the force. Thin-wall ultracentrifuge tubes are held in shape by the liquid in them and buckle if run part-full.
- Disturbing the pellet on the way out. A soft debris pellet lifts as soon as the tube is tipped, putting back what the spin removed.
Key terms
Practise these as flashcards →Relative centrifugal field: the outward acceleration as a multiple of gravity, 1.118e-5 × r(cm) × rpm². The quantity that transfers between rotors.
A particle's sedimentation velocity per unit field, in units of 10⁻¹³ s. A property of the particle, so 80S means the same thing in any rotor.
2.53e11 × ln(rmax/rmin) / rpm²: the hours a 1 S particle needs to cross the tube in that rotor at that speed. A species of s svedbergs clears in k/s hours.
A gradient run stopped while bands are still migrating, so position reports sedimentation rate (size and shape).
A gradient run taken to equilibrium, so position reports buoyant density and is independent of how long you spin.
The radius of the bottom and the top of the liquid column. Force is quoted at rmax; the path length between them sets the run time.
Reducing a rotor's maximum speed for dense samples or for accumulated stress cycles, as the rotor manual specifies.
When to use it
- Clearing a lysate, a culture or a digest of cells, debris and precipitate before chromatography, a pull-down or a gel.
- Harvesting cells or bacteria from medium, and washing a pellet by resuspension and re-pelleting.
- Concentrating a precipitate, after an alcohol or ammonium sulfate precipitation the pellet only exists because you spun it down.
- Separating subcellular fractions, viruses or ribonucleoprotein complexes by sedimentation rate or buoyant density.
Compared with alternatives
Differential and gradient centrifugation applied systematically to organelles, with the marker assays that tell you which fraction is which.
Prefer filtration when you want to remove particles by size rather than by sedimentation rate, or to sterilise rather than to separate.
Use AUC when the sedimentation coefficient itself is the measurement (oligomeric state, aggregation) rather than a means of separation.
A spin concentrates and separates; dialysis or a desalting column is what changes the buffer the sample is in.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A fixed-angle benchtop rotor taking 1.5–2 mL tubes, typically reaching 15,000–21,000 × g. The everyday workhorse for clearing lysates and digests, collecting alcohol precipitates, washing small pellets and spinning columns. Short path length means pellets form in minutes, but the modest force means anything slower than a few thousand svedbergs stays in the supernatant.
- Fixed-angle: the pellet forms on the tube wall at an angle, so take the supernatant off from the opposite side.
- Radii differ widely between models, so the same rpm is a different force: check the rotor's rmax before copying a speed.
- Refrigerated models need pre-cooling: the first minutes of an unprecooled run happen at room temperature.