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Protocol for Differential Velocity Centrifugation

Principles of Differential Velocity Centrifugation

Differential velocity centrifugation is a technique used to separate particles or components in a sample based on their different sedimentation velocities. It relies on the principle that particles of different sizes and densities will sediment at different rates when subjected to centrifugal force. This technique is commonly employed in the field of biotechnology and biochemistry for the isolation and purification of organelles, subcellular fractions, and macromolecules.

The process involves subjecting the sample to centrifugal force, which causes the particles to sediment based on their size and density. By carefully controlling the centrifugation conditions, such as rotor speed, duration, and temperature, it is possible to separate components of interest from the rest of the sample. The resulting fractions can then be further processed or analyzed.

Applications of Differential Velocity Centrifugation

Organelle Isolation: One of the primary applications of differential velocity centrifugation is the isolation of specific organelles from complex cellular mixtures. By adjusting the centrifugation parameters, such as rotor speed and duration, organelles such as mitochondria, nuclei, lysosomes, and microsomes can be separated and purified.

Subcellular Fractionation: This technique is widely used for subcellular fractionation, which involves separating different cellular components based on their sedimentation rates. It allows researchers to isolate and study specific subcellular structures or compartments, such as the rough endoplasmic reticulum, Golgi apparatus, or plasma membrane.

Macromolecule Purification: Differential velocity centrifugation is also utilized for the purification of macromolecules, such as proteins, nucleic acids, and ribosomes. By optimizing the centrifugation conditions, it is possible to separate these macromolecules from other cellular components or contaminants, facilitating further analysis or downstream applications.

Virus Purification: The technique is employed in virology for the purification of viruses from infected cell cultures or biological samples. By subjecting the sample to differential velocity centrifugation, viruses can be separated from host cell debris and other contaminants, enabling their characterization and study.

Particle Size Analysis: Differential velocity centrifugation can be utilized for particle size analysis and characterization. By subjecting a sample containing particles of varying sizes to centrifugation, the sedimentation rates can provide information about the size distribution of the particles present in the sample.

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Materials Required of Differential Velocity Centrifugation

  • Rats that have not been fed overnight and have been recently sacrificed
  • Homogenization solution: A solution containing 0.25 to 0.3 M sucrose with optional additives (refer to the recipe; if PMSF is included, add it just before use)
  • Motorized pestle with a high-torque motor (e.g., laboratory stirrer, VWR or Fisher)
  • Cheesecloth
  • Tubes suitable for low-speed centrifugation: 1.5-ml microcentrifuge tubes (for small-scale preparation) or 15- or 50-ml graduated screw-cap tubes (for larger-scale preparations)
  • Low-speed centrifuge capable of generating a centrifugal force of 600 × g at 4◦C
  • Teflon-glass homogenizer (A.H. Thomas or equivalent) of an appropriate size for the scale of the experiment: type AA (1 ml) to type C (40 ml)
  • 200-μm mesh nylon screen (Tetko)
  • Dounce-type glass homogenizer
  • Polycarbonate or polyallomer tubes suitable for ultracentrifugation (e.g., 3-ml size to fit Beckman TLA-100.3 rotor) or polycarbonate bottle assemblies (e.g., 10-ml size to fit Beckman 50Ti rotor or 25-ml size to fit Beckman 60Ti or 70Ti rotor)
  • Ultracentrifuge and fixed-angle rotor appropriate for the scale of the experiment (e.g., Beckman 50Ti, 60Ti, or 70Ti) or tabletop ultracentrifuge and rotor (e.g., Beckman tabletop with TL100 rotor)

NOTE: Prepare all solutions using Milli-Q-purified water or an equivalent.

NOTE: Perform all operations, starting from tissue removal and weighing, at either 0◦C (in an ice bucket) or 4◦C (in a cold room).

Procedure of Differential Velocity Centrifugation

Homogenization of the Sample:

1. Take the liver from a freshly sacrificed rat that has undergone an overnight fast (to deplete hepatic glycogen). Keep the liver on ice as much as possible. Weigh the liver and then roughly mince it using scissors or a razor blade in a weighing boat. It is not recommended to use stored frozen tissues for organelle purification procedures.

2. Homogenize the minced liver at a concentration of 20% (w/v) in the homogenization medium. Use a motor-driven pestle with a high-torque motor and perform five up-down strokes at 1700 rpm.

3. Filter the homogenate through four layers of cheesecloth to remove any solid particles.

Low-Speed Centrifugation to Remove Nuclei and Larger Particulates:

4. Transfer the homogenate to tubes suitable for low-speed centrifugation. Centrifuge the tubes at 600 × g (1700 rpm for a 20-cm-radius rotor) for 10 minutes at 4◦C. For small-scale procedures, you can perform this centrifugation in 1.5-ml microcentrifuge tubes at 10,000 rpm (6600 × g) for 40 seconds.

5. For tissues like the pancreas and salivary gland: Take the pellet from the previous step and resuspend it in the original volume of homogenization medium. Rehomogenize the suspension with a Teflon-glass pestle and centrifuge again under the same conditions. Combine the resulting supernatant with the supernatant from the first centrifugation. Filter this combined supernatant through a 200-μm mesh nylon screen and disperse it with 3 to 4 strokes in a Dounce homogenizer. It is important to note that pancreas and salivary gland (and some other tissues) are more difficult to homogenize than the liver. To ensure successful organelle purification, it is recommended to employ the two-step homogenization procedure when appropriate.

6. The pellet obtained after centrifugation is the crude nuclear fraction, and the supernatant (or combined supernatants, for optional two-step homogenization) is the postnuclear supernatant (PNS) fraction. Remove any accumulated lipid from the surface of the PNS and withdraw the PNS, taking care not to disturb the pellet. Transfer the PNS to polycarbonate tubes suitable for ultracentrifugation.

Preparation of Mitochondrial and Microsomal Fractions by Successive Ultracentrifugations:

7. Centrifuge the PNS at 10,000 × g (e.g., 10,000 rpm in Beckman 50Ti, 60Ti, or 70Ti) for 30 minutes at 4◦C to pellet the mitochondrial fraction.

8. Remove the supernatant and transfer it to clean polycarbonate ultracentrifuge tubes.

9. Resuspend the pellet enriched with mitochondria and transfer it to a Dounce homogenizer. Disperse the pellet into a fine suspension by performing several manual strokes. It is important to note that the pellet also contains lysosomes, peroxisomes (which are less abundant), and small amounts of rough and smooth microsomes that happened to pellet.

10. Optionally, for further purification of mitochondria and separation from smaller-sized contaminants, you can repeat steps 6 and 7. This approach has been used by Carvalho-Guerra (1974) to prepare liver mitochondrial fractions.

11. Centrifuge the supernatant obtained from step 7 at 100,000 × g for 2 hours at 4◦C using a fixed-angle rotor.

12. The resulting pellet is the total microsomal fraction, consisting of vesicles derived from the rough endoplasmic reticulum and various smooth-membrane-bounded organelles. It may also contain fragments from mitochondria and larger-sized organelles that were damaged during homogenization. Remove the postmicrosomal (or "high-speed") supernatant, which is generally used as "cytosol" in various assays.

13. Resuspend the microsomal pellet in an appropriate buffer for assays or additional purification. Disperse the pellet using a Dounce homogenizer and store it at 4◦C until further use.

* For Research Use Only. Not for use in diagnostic procedures.
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