Differential Centrifugation

Core Concept

Differential centrifugation separates the components of a cell homogenate by spinning at progressively higher speeds, pelleting first the largest and densest structures, then smaller ones. At each step, the pellet (sedimented material) and the supernatant (remaining liquid) are separated; the pellet is enriched in one organelle class and the supernatant carries everything smaller. The technique is a workhorse of cell biology because it lets researchers obtain fractions enriched in nuclei, mitochondria, membranes, or ribosomes for biochemical study. Its crucial limitation: each fraction is an enrichment, not a purification — it is contaminated with other organelles of similar size/density.

Key Components

• Centrifuge/rotor: spins tubes to generate centrifugal force on particles, pulling them outward and downward to the tube bottom.
• Centrifugal force (relative centrifugal force, RCF): RCF = 1.12 × 10⁻⁵ × r × (rpm)², where r = rotor radius (cm) and rpm = revolutions per minute. Expressed in units of g (multiples of Earth's gravity).
• Homogenate: cells broken open (homogenized) gently in an isotonic buffer, rupturing the plasma membrane but leaving organelles intact.
• Pellet: the material that sediments to the bottom of the tube.
• Supernatant: the liquid left above the pellet, containing the unsedimented (smaller/lighter) material.
• Marker enzymes: enzymes known to be unique to a particular organelle, used to confirm which organelle a fraction contains (e.g., succinate dehydrogenase for mitochondria, catalase for peroxisomes, acid phosphatase for lysosomes).

Mechanism / How It Works

1. Homogenize. Break cells open in a cold, isotonic buffer to release intact organelles (nuclei, mitochondria, lysosomes, ER fragments called "microsomes," ribosomes) into a suspension.
2. Low-speed spin (~600–1,000 × g, ~10 min). The largest, densest structures — nuclei, unbroken cells, and debris — pellet. The supernatant is saved (contains everything else).
3. Medium-speed spin (~10,000–15,000 × g). Mitochondria (and often lysosomes and peroxisomes) pellet from the previous supernatant. This "mitochondrial fraction" is enriched but contaminated.
4. High-speed spin (~100,000 × g, "ultracentrifugation"). The microsomal fraction (fragmented endoplasmic reticulum and plasma membrane) pellets; the remaining supernatant contains cytosol (soluble proteins).
5. Very high-speed spin (~150,000–200,000 × g). Ribosomes and large macromolecular complexes can be pelleted.
6. Validation. Each fraction is tested with marker enzymes to identify and assess contamination. The sedimentation of a particle depends on its size, shape, and density, and its sedimentation coefficient (Svedberg units, S), not merely its mass.

Energy and Directionality

The driving force is mechanical, not metabolic: the centrifuge converts electrical energy into rotation, and the resulting centrifugal field does work on particles. The directionality is physical — particles always move outward (down the tube) at a rate set by the centrifugal force, with larger/denser particles sedimenting faster. No ATP/GTP is consumed by the organelles; the "energy" spent is the machine's. This is why buffers are kept cold and isotonic: to preserve the organelles' own (ATP-powered) integrity during the procedure.

Technique (How It Is Done)

• Homogenization buffer: isotonic (e.g., 0.25 M sucrose) and cold, with protease inhibitors and ions to keep organelles intact.
• Sequential spins: pellet → save pellet on ice; transfer supernatant to a fresh tube for the next, faster spin.
• Density-gradient centrifugation (for higher purity): layer the crude fraction on a density gradient (e.g., sucrose or Percoll); spin to equilibrium, and organelles band where their density matches the medium. This separates organelles by density, overcoming the overlap that plagues differential centrifugation (e.g., separating lysosomes from mitochondria).
• Validation: assay marker enzymes and, in modern work, mass-spectrometry proteomics or microscopy to confirm fraction identity and purity.
• Key caveat to state explicitly: differential centrifugation enriches fractions; it does not purify them. Contamination and organelle damage (e.g., ER fragmentation into microsomes) are expected.

How it works

1. Homogenize. Break cells open in a cold, isotonic buffer to release intact organelles (nuclei, mitochondria, lysosomes, ER fragments called "microsomes," ribosomes) into a suspension.
2. Low-speed spin (~600–1,000 × g, ~10 min). The largest, densest structures — nuclei, unbroken cells, and debris — pellet. The supernatant is saved (contains everything else).
3. Medium-speed spin (~10,000–15,000 × g). Mitochondria (and often lysosomes and peroxisomes) pellet from the previous supernatant. This "mitochondrial fraction" is enriched but contaminated.
4. High-speed spin (~100,000 × g, "ultracentrifugation"). The microsomal fraction (fragmented endoplasmic reticulum and plasma membrane) pellets; the remaining supernatant contains cytosol (soluble proteins).
5. Very high-speed spin (~150,000–200,000 × g). Ribosomes and large macromolecular complexes can be pelleted.
6. Validation. Each fraction is tested with marker enzymes to identify and assess contamination. The sedimentation of a particle depends on its size, shape, and density, and its sedimentation coefficient (Svedberg units, S), not merely its mass.

Common confusions

• "Differential centrifugation purifies organelles." — Wrong. It enriches them; each pellet is contaminated with other organelles of similar sedimentation properties.
• "The first pellet is pure nuclei." — No. It contains nuclei plus unbroken cells and large debris.
• "Higher speed always separates smaller from larger perfectly." — Separation is by sedimentation rate (size, shape, AND density), and overlap between organelle classes is routine — hence the need for density gradients.
• "The supernatant is waste." — Wrong. The supernatant is where the smaller/unsedimented material is; it is often the fraction of interest (e.g., cytosol).
• "rpm alone specifies a spin." — No. Centrifugal force depends on rotor radius too; protocols should state g (RCF), not just rpm.

Quick review

• Differential centrifugation: sequential spins at increasing g → pellets by size/density.
• RCF (g) = 1.12 × 10⁻⁵ × r × rpm².
• Order: nuclei → mitochondria/lysosomes → microsomes → ribosomes (with cytosol in the final supernatant).
• Pellet vs. supernatant at each step.
• Fractions are ENRICHED, not pure.
• Validate with marker enzymes; density gradients improve purity.