Lecture 9: Cell Fractionation

Cell Fractionation

Learning Objectives

• Understand cell fractionation.
• Outline the cell fractionation procedure.
• Differentiate between differential centrifugation, velocity sedimentation, and equilibrium sedimentation.

What is Cell Fractionation?

• Cell fractionation is a technique used to study organelles and macromolecules, which is difficult to do in whole cells.
• It's a useful technique for studying cell structure and the function of cellular structures.
• Cell fractionation allows researchers to prepare specific cell components and identify their functions.
  • Example: Biochemical tests on mitochondria fractions show the presence of respiratory enzymes, determining mitochondria as the site of cellular respiration.

Process of Cell Fractionation

• Involves breaking cells apart and separating organelles and other subcellular structures.
• Uses a centrifuge.
• The principle is to spin samples at different speeds to isolate the cell organelles/molecules.
• Samples are placed in tubes within a centrifuge.
• After centrifugation, a pellet forms at the bottom of the tube.

Purpose of Cell Fractionation

Protein Enrichment

• Enrich target proteins and improve the detection of low abundance proteins.
• Concentrates particular proteins in a biological sample for further analysis and identification.

Protein Characterization

• Identify the subcellular localization of a protein.
• Characterize its structure and function.

Protein Translocation

• Monitor translocation of cell signaling molecules from the cytoplasm to the nucleus.
• Understand how proteins move between cellular compartments.

Cell Fractionation Steps

1. Homogenization
2. Centrifugation

Homogenization

• Breaks up tissue by mechanical force (blender, pestle/mortar, or homogenizer) to release organelles.
• Breaks open the cell wall or cell membrane.

• Lysis: When the inner contents of a cell leak out into the environment.
• Homogenization can be done mechanically or chemically:
  • Mechanical Breakage: Cell solution placed in a high-speed blender and then filtered.
  • Chemical Homogenization: Enzymes or detergents added to the solution to dissolve the lipid bilayer.
• Once the cell solution is homogenized, organelles leak out due to lysis, and the solution becomes a homogenate.
• The homogenate is transferred to centrifuge test tubes, capped, and placed in a centrifuge.

Homogenization Solution

Sample Must Be:

• Cold
• Isotonic
• pH buffered

Centrifugation

• Centrifugation is a technique for separating substances.
• Commonly used in laboratories for separating biological molecules from a crude extract.
• A centrifuge is a high-speed spinning device that separates the components of a solution into layers, called fractions.
• Particles are separated from a solution according to their size, shape, density, the viscosity of the medium, and rotor speed.
• It has two main components: an electric motor to spin and a rotor to hold the tubes.

How Centrifugation Works

• In a solution, particles whose density is higher than that of the solvent sink and form a sediment, which is the PELLET.
• Particles that are lighter float to the top, which is the SUPERNATANT.
• The supernatant is separated from the pellet by decantation or collected using a pipette.
• The greater the difference in density, the faster they move.
• At relatively low speed, large components such as nuclei sediment to form a pellet.
• At slightly higher speed, a pellet of mitochondria is deposited.
• At even higher speeds and with longer periods of centrifugation, first vesicles and then ribosomes can be collected.

Uses of Centrifugation

• Concentration of cells from large volumes of fluids.
  • Cells grown in liters of culture medium can be sedimented from large volumes of fluid and re-suspended in a much smaller volume.
• To separate cellular components.

Types of Centrifuges

1. Low-speed table top centrifuge
2. High-speed centrifuge
3. Ultracentrifuge
4. Microcentrifuge

Low Speed Centrifuge

• Most commonly used in labs.
• Not temperature controlled (operates at room temperature).
• Maximum speed of 4000-5000 rpm.

Ultracentrifuge

• An ultracentrifuge consists of a refrigerated, low-pressure chamber containing a rotor driven by an electrical motor, capable of high-speed rotation.
• Samples are placed in tubes within or attached to the rotor.
• Ultracentrifuges are advanced centrifuges that separate smaller molecules that cannot be separated by traditional centrifuges.
• The speed of these centrifuges can reach as high as 150,000 rpm.

Types of Centrifugation

1. Differential centrifugation
2. Velocity sedimentation
3. Equilibrium sedimentation

Differential Centrifugation

• Differential centrifugation is a procedure used in cell biology to separate certain organelles from whole cells for further analysis.
• Separation is based on size, with larger and denser particles pelleting at lower centrifugal forces.
• Repeated centrifugation at progressively higher speeds will fractionate cell homogenates into their components.
• The larger and denser components experience the greatest centrifugal force and move most rapidly, sedimenting to form a pellet at the bottom of the tube.
• The smaller, less dense components remain in suspension above (the supernatant).

Differential Centrifugation Process

1. A tissue sample is first homogenized to break the cell membranes and mix up the cell contents.
2. The homogenate is then subjected to repeated centrifugations, each time removing the pellet and increasing the centrifugal force.
3. The desired layer is extracted for further analysis.

Velocity Sedimentation

• A finer degree of separation can be achieved by layering the homogenate in a thin band on top of a gradient solution that fills a centrifuge tube.
• When centrifuged, the various components in the mixture move as a series of distinct bands through the solution, each at a different rate.
• For the procedure to work effectively, the bands must be protected from convective mixing.
• The solution contains a continuous shallow gradient of sucrose that increases in concentration toward the bottom of the tube.
• The resulting density gradient has the dense end at the bottom of the tube, keeping each region of the solution denser than any solution above it, thereby preventing convective mixing.
• When sedimented through sucrose gradients, different cell components separate into distinct bands that can be collected individually.
• The gradient is typically 5-20% sucrose.
• After an appropriate centrifugation time, the bands may be collected, most simply by puncturing the plastic centrifuge tube and collecting drops from the bottom.

Velocity Sedimentation Process

1. A density gradient of a medium is created by gently laying the lower concentration over the higher concentrations in a centrifuge tube.
2. The sample is then placed over the gradient, and the tubes are placed in an ultracentrifuge.
3. The particles travel through the gradient until they reach a point at which their density matches the density of the surrounding medium.
4. The fractions are removed and separated, obtaining the particles as isolated units.

Equilibrium Sedimentation

• The ultracentrifuge is also used to separate cell components based on their buoyant density, independently of their size and shape.
• This process is sensitive enough to separate macromolecules that have incorporated heavy isotopes from the same macromolecules that contain lighter, common isotopes.
• The method is also called density gradient centrifugation.

Equilibrium Sedimentation

• In this case, the sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or caesium chloride.
• Each cell component begins to move down the gradient but eventually reaches a position where the density of the solution is equal to its own density.
• At this point, the component floats and can move no farther.
• A series of distinct bands is thereby produced in the centrifuge tube, with the bands closest to the bottom of the tube containing the components of highest buoyant density.