Centrifugation and Cell Fractionation Study Notes

Principles and Mechanics of Centrifugation

• Definition: A separation technique for particles in liquids based on their size, shape, density, and medium viscosity using centrifugal force.
• Mechanics: High-speed rotation generates force perpendicular to the axis, causing denser particles to move outward and sediment as a pellet.
• Sedimentation Rule: The sedimentation rate is zero when the density of the particle and its surrounding medium are equal.
• Factors Affecting Rate:
  • Particle size and density (directly proportional).
  • Medium density and viscosity (inversely proportional).
  • Rotor speed and radius ($r$).
  • Temperature (high temperatures may damage biological samples).

Types of Centrifuges

• Low speed centrifuge: Maximum speed of $6,000\text{ rpm}$; used for routine sedimentation of heavy particles like red blood cells.
• High Speed Centrifugation: Speed range of $6,000 - 25,000\text{ rpm}$; features temperature control for bacteria and organelle separation.
• Ultra Centrifuge: Speed range of $25,000 - 100,000\text{ rpm}$ or more; requires refrigeration and high vacuum to manage intense heat. Used for both preparative and analytical work.

Centrifugation Methodologies

• Differential Centrifugation:
  • Separates components based on differing sedimentation rates through stepwise increases in centrifugal field.
  • Sequential Separation:
    • $600\,xg$: Nuclei and cell debris.
    • $10,000\,xg$: Mitochondria, lysosomes, and peroxisomes.
    • $20,000 - 40,000\,xg$: Membrane fragments.
    • $100,000\,xg$: Ribosomes and microsomes.
    • Supernatant: Cytosol (soluble proteins).
• Density Gradient Centrifugation:
  • Rate-zonal: Separates particles based on size and mass using media like sucrose. Centennial duration must be limited to prevent all particles from pelleting.
  • Isopycnic (Equilibrium): Separates solely by density. Particles migrate to a point in the gradient equal to their own density (equilibrium point). Used for DNA, RNA, and lipoproteins.

Cell Fractionation Steps and Lysis Methods

• 1. Tissue Preparation: Samples are washed with isotonic buffer to maintain osmotic balance.
• 2. Homogenization: breaking cell membranes to create a homogenate.
  • Mechanical: Mortar and pestle or grinding.
  • Ultrasonic (Sonification): Uses high-frequency sound waves; fast but generates heat.
  • French Press: High-pressure valve system; preserves organelles well.
  • Osmotic Lysis: Uses hypotonic solutions to burst animal cells.
  • Enzymatic Lysis: Uses lysozyme (bacteria), cellulase (plants), or chitinase (fungi).
  • Chemical Lysis: Uses detergents like Sodium Dodecyl Sulfate ($SDS$) or Triton X114.
• 3. Filtration: Removes unbroken cells and large debris.
• 4. Separation: Sequential centrifugation to isolate specific organelles.

Applications and Safety

• Molecular Biology: DNA extraction, RNA isolation, and PCR preparation.
• Clinical/Biochemistry: Blood analysis, serum preparation, and protein purification.
• Precautions:
  • Balance tubes by equal volume and weight.
  • Verify rotor installation and lock lid before operation.
  • Use sealed buckets for infectious materials.
  • Maintain equipment regularly and avoid improper balancing to prevent damage.