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Differential centrifugation
Differential centrifugation is a technique used to separate cellular components like organelles based on sedimentation rate differences caused by varying density, size, shape when spun at increasing centrifugal forces; it involves homogenizing a sample, centrifuging sequentially at low speeds to pellet larger components then higher speeds to further separate smaller ones, allowing fractionation of components from nuclei to ribosomes into pellets and supernatants. Differential centrifugation has applications in separating various mixtures and purifying biomolecules, cells, and subcellular structures.
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Differential centrifugation
- 1. Differential centrifugation Dr. P.Suganya Assistant Professor Department of Biotechnology Sri Kaliswari College (Autonomous) Sivakasi
- 3. • Differential centrifugation(also known as differential velocity centrifugation) is a common procedure in biochemistry and cell biology, which is used to separate organelles and other sub-cellular particles based on their sedimentation rate.
- 4. Principle • In aviscous fluid, the rate of sedimentation of a given suspended particle (as long as the particle is more dense than the fluid) is largely a function of the following factors: • Gravitational force • Difference in density • Fluid viscosity • Particle size and shape
- 5. Procedure • Differential centrifugationcan be used with intact particles (e.g. biological cells, microparticles, nanoparticles), or used to separate the component parts of a given particle. • Using the example of a separation of eukaryotic organelles from intact cells, the cell must first be lysed and homogenized (ideally by a gentle technique, such as dounce homogenization; harsher techniques or over homogenization will lead to a lower proportion of intact organelles). • Once the crude organelle extract is obtained, it may be subjected to a varying centrifugation speeds to separate the organelles:
- 6. Typical differential centrifugationparameters for a biological sample Sample input G force Time Instrument needed Pellet contents Supernatant contents Unlysed (eukaryotic) cells 100 x g 5 min Benchtop fixed- angle centrifuge, or swinging bucket centrifuge Intact (eukaryotic) cells, macroscopic debris Varies depending on sample Gently lysed cells (e.g. dounce homogenizer) 600 x g 10 min Benchtop fixed- angle centrifuge, or swinging bucket centrifuge Nuclei Cytosol, non- nuclei organelles Supernatant of previous row 15,000 x g 20 min Benchtop fixed- angle centrifuge Mitochondria, chloroplasts, lysosomes, peroxisomes Cytosol, microso mes (known as post mitochondrial supernatant) Supernatant of previous row 50,000 x g - 100,000 x g 60 min High speed fixed-angle centrifuge, or vacuum ultracentrifuge Plasma membrane, microsomal fraction, large polyribosomes Cytosol, ribosomal subunits, small polyribosomes, enzyme complexes Supernatant of previous row 50,000 x g - 100,000 x g 120 min Vacuum ultracentrifug e Ribosomal subunits, small poly ribosomes, some soluble enzyme complexes Cytosol
- 7. • It isthe most common type of centrifugation employed. • Tissue such as the liver is homogenized at 32 degrees in a sucrose solution that contains buffer. • The homogenate is then placed in a centrifuge and spun at constant centrifugal force at a constant temperature. • After some time a sediment forms at the bottom of a centrifuge called pellet and an overlying solution called supernatant. • The overlying solution is then placed in another centrifuge tube which is then rotated at higher speeds in progressing steps.
- 8. Applications • To separatetwo miscible substances • To analyze the hydrodynamic properties of macromolecules • Purification of mammalian cells • Fractionation of subcellular organelles (including membranes/membrane fractions) Fractionation of membrane vesicles • Separating chalk powder from water • Removing fat from milk to produce skimmed milk • Separating particles from an air-flow using cyclonic separation • The clarification and stabilization of wine • Separation of urine components and blood components in forensic and research laboratories • Aids in the separation of proteins using purification techniques such as salting out, e.g. ammonium sulfate precipitation.