Cell fractionation is a fundamental laboratory technique used to isolate specific cellular components, such as organelles and macromolecules, for detailed study. This process allows scientists to determine the function, composition, and location of cellular structures without interference from the complex environment of the whole cell. The overall purpose is to gently break the cell open and then separate the released contents based on physical properties like size, density, and shape. Isolating individual cellular compartments enables researchers to perform highly specific biochemical and molecular analyses, gaining insights into biological machinery.
Breaking Open the Cell
The first stage of cell fractionation involves the controlled disruption of the cell boundary, known as homogenization or cell lysis. This step must be performed carefully to rupture the outer plasma membrane or cell wall while preserving the structural integrity and functionality of internal organelles. Cells are typically suspended in an isotonic buffer solution to prevent osmotic damage, and the entire process is conducted at low temperatures to minimize the breakdown of components by cellular enzymes.
Mechanical homogenization is a common physical method for disruption, involving the application of shear force to the cell suspension. Devices like the Dounce homogenizer are frequently used, consisting of a glass pestle driven into a glass tube with a narrow clearance. The shearing force generated tears open the plasma membrane, allowing cellular contents to spill out while leaving larger, more robust organelles, such as the nucleus, relatively undamaged.
Another technique is sonication, which utilizes high-frequency ultrasonic waves to create rapid pressure changes in the liquid medium. These pressure waves generate microscopic cavitation bubbles that collapse, producing shock waves that effectively lyse the cells. Sonication offers precise control but generates significant heat, so it must be performed in short bursts on ice to prevent protein denaturation. For resilient cell types, such as bacteria, a French press may be employed, which forces the cell suspension through a tiny orifice under extremely high pressure to achieve lysis.
Separating Components Using Centrifugal Force
Once the cells are broken open, the resulting mixture, known as the homogenate, is subjected to differential centrifugation to separate the contents. This technique relies on the principle that particles suspended in a liquid medium will sediment at a rate determined by their size, density, and the force applied. By performing sequential spins at progressively increasing speeds, scientists can selectively pellet out cellular components from the liquid supernatant.
The process begins with a relatively low-speed spin, typically around \(1,000 times g\) for a short duration. This initial centrifugation causes the largest and densest particles to sediment quickly, forming a compact pellet. This pellet primarily contains intact cells, large cell debris, and cell nuclei. The remaining liquid, or supernatant, which holds the smaller components, is then carefully removed and transferred to a new tube.
The second spin is performed at a higher g-force, often in the range of \(10,000\) to \(20,000 times g\), to sediment the next heaviest set of organelles. This fraction typically includes medium-sized organelles, such as mitochondria, lysosomes, and peroxisomes, which form the second pellet. The supernatant from this step is collected and centrifuged at an even higher force, sometimes up to \(100,000 times g\) or more, using an ultracentrifuge.
The high-speed spin pellets the smaller membrane-bound vesicles and fragments, commonly referred to as microsomes, derived from the fragmented endoplasmic reticulum and Golgi apparatus. Finally, the remaining supernatant, which contains the smallest soluble molecules, proteins, and the cell’s fluid matrix (the cytosol), is isolated. This sequential increase in centrifugal force allows for the effective separation of cellular contents, providing distinct fractions for individual analysis.
Confirming What Was Isolated
After separation, scientists must confirm the purity and identity of the isolated fractions to ensure the success of the protocol. Since a fraction is rarely perfectly pure and often contains trace contamination, verification is a necessary measure of quality control. This confirmation is achieved through specific biochemical assays that detect marker enzymes.
Marker enzymes are proteins uniquely localized to a specific organelle, acting as biochemical identifiers. For instance, researchers confirm the mitochondrial fraction by looking for the activity of succinate dehydrogenase, an enzyme exclusively found in the inner mitochondrial membrane. High activity of this enzyme in the mitochondrial pellet and low activity elsewhere indicates successful isolation. Acid phosphatase is used as a marker for the lysosome fraction, and lactate dehydrogenase serves as a reliable marker for the cytosolic fraction. By measuring the specific activity of these enzymes across all collected fractions, researchers can quantitatively assess the degree of enrichment and the level of cross-contamination.
Practical Research Applications
Isolating specific cellular components provides a powerful platform for a wide range of biological and medical investigations. By separating the cell into its working parts, researchers can study complex processes in a more simplified environment. One application is determining the precise localization of a protein of interest to establish its function. For example, if a newly discovered protein is highly concentrated in the mitochondrial fraction, it suggests a function related to cellular energy production or metabolism.
Fractionation is frequently employed to study how disease progression affects specific organelles, such as tracking mitochondrial dysfunction in neurodegenerative disorders like Parkinson’s disease. Researchers isolate mitochondria from diseased cells to analyze changes in their protein composition or enzyme activity. The technique also holds value in the development of new therapeutics, particularly in tracking drug delivery mechanisms at a subcellular level. Scientists use isolated fractions to observe how a drug interacts with a specific organelle, like the lysosome, to assess its effectiveness or potential toxicity.