Cell fractionation is a fundamental laboratory technique used to separate the complex mixture of a cell into its distinct, usable parts. The primary goal is to isolate specific internal structures, such as organelles or the surrounding fluid, so their individual functions can be studied without interference from other cellular components. By obtaining purified fractions, scientists gain a controlled environment to analyze biochemical processes, metabolic pathways, and the molecular machinery of life outside the crowded context of the intact cell. This approach has been foundational in cellular biology for demonstrating where various biochemical activities, like energy production or protein synthesis, occur within the cell.
Breaking Down the Cell Structure
Before separation, cells must first be broken open, a preparatory step known as homogenization or lysis. This process is carefully controlled to disrupt the outer cell membrane or cell wall, releasing the internal contents while preserving the integrity and function of the organelles. Cells or tissues are typically suspended in an isotonic buffer solution, often containing sucrose, which helps maintain the correct osmotic pressure to prevent the organelles from swelling or shrinking.
Mechanical methods, such as grinding, applying high pressure, or using an instrument that shears the cells, are employed to achieve this rupture. The resulting mixture of released cell components, unbroken cells, and surrounding buffer is referred to as the cellular homogenate. This crude homogenate is then ready for the next stage of separation.
The Mechanics of Separation
The technique most commonly used for separation is differential centrifugation, which separates components based on differences in their size and density. This method involves subjecting the homogenate to a series of increasing centrifugal forces applied over specific durations. The underlying principle is that the gravitational force exerted by the centrifuge causes particles to sediment at rates proportional to their mass and density.
Initially, the homogenate is spun at a relatively low speed, such as 600 times the force of gravity (600 x g), for a short time. This gentle force causes only the largest and densest components, primarily the intact nuclei and any remaining whole cells, to settle out and form a pellet. The remaining liquid, called the supernatant, is then carefully decanted into a new tube for the next cycle.
The next spin is performed at a higher force, perhaps 15,000 x g, which pellets the next heaviest fraction, typically consisting of mitochondria, lysosomes, and peroxisomes. This iterative process continues, with each successive increase in speed and duration isolating progressively smaller components. The smaller, membrane-bound vesicles derived from the endoplasmic reticulum and Golgi apparatus, collectively termed the “microsomal fraction,” are pelleted at much higher forces (around 80,000 x g). Finally, the last supernatant is the cytosol, containing the soluble proteins and macromolecules of the cell.
Analyzing the Isolated Components
The value of cell fractionation lies in the ability to study the function and composition of each isolated fraction. Once separated, the purified fractions, such as isolated mitochondria or the cytosolic fluid, can be subjected to detailed biochemical analysis. For example, researchers can measure the rate of ATP production within an isolated mitochondrial fraction to understand cellular respiration, or they can study protein synthesis using the isolated ribosomes and enzymes found in the cytosolic fraction.
This compartmentalized analysis is instrumental in understanding disease mechanisms by determining the precise location of molecules. If a disease involves the buildup of a specific protein, cell fractionation can isolate the nuclear, membrane, or cytoplasmic fractions to pinpoint where that protein is accumulating. Isolated organelles are also used for proteomics studies, allowing scientists to identify the complete set of proteins associated with a specific compartment, revealing new insights into organelle-specific functions and signaling pathways.