Differential ultracentrifugation is a laboratory method used to separate particles in a liquid mixture based on their size and density. The technique involves spinning samples at very high speeds, generating powerful forces that cause components to separate. This procedure allows for the purification of materials such as cellular organelles, viruses, and large molecules like proteins or DNA. By separating these components, researchers can study their specific functions and structures in isolation, making it a foundational first step in many experimental workflows.
The Separation Process
The process begins with cell fractionation, where cells are broken open using a homogenizer. This device acts like a blender to break cell membranes and release the internal contents into a solution. This initial mixture, known as a homogenate, is prepared in a cold, buffered, and isotonic solution to prevent damage to the organelles from temperature changes, pH fluctuations, or osmosis.
This homogenate is then subjected to a series of centrifugation steps at progressively increasing speeds. In the first and slowest spin, the largest and densest components, such as whole cells that were not broken, the nuclei, and the cytoskeleton, are forced to the bottom of the tube. This solid material collected at the bottom is called the pellet. The remaining liquid, which contains smaller particles, is called the supernatant.
This supernatant is carefully removed and transferred to a new tube for the next stage. It is then spun at a significantly higher speed, generating a greater centrifugal force. This second step pellets medium-sized components, such as mitochondria, lysosomes, and peroxisomes. The supernatant is collected again, and the process is repeated.
Each subsequent step uses a higher centrifugation speed to pellet progressively smaller particles. For instance, a third spin at an even higher force can pellet microsomes, which are fragments of the endoplasmic reticulum. A final, very high-speed spin can separate the smallest components, like ribosomes and large macromolecules. This sequential separation based on increasing centrifugal force is the defining characteristic of the method.
Key Principles of Sedimentation
An ultracentrifuge generates extreme centrifugal forces by spinning samples at speeds that can exceed 100,000 revolutions per minute (rpm). These forces are hundreds of thousands of times greater than Earth’s gravity (g-force). This force compels particles suspended in a liquid to move away from the center of rotation.
A particle’s sedimentation rate is determined by its size, shape, and density. Larger or denser particles will move faster and pellet at lower speeds, while smaller, less dense particles require greater force to separate. The density and viscosity of the medium in which the particles are suspended also influence this rate.
This sedimentation rate is scientifically quantified using the Svedberg unit (S), which measures how quickly a particle sediments under a specific force. For example, ribosomes are often distinguished by their Svedberg values, such as the 70S ribosome found in prokaryotes, which is composed of 50S and 30S subunits.
Applications in Scientific Research
Differential ultracentrifugation is widely used for isolating subcellular organelles from a cell lysate. This allows scientists to create purified fractions of nuclei, mitochondria, or ribosomes. By separating these organelles, researchers can conduct experiments to understand their specific biological roles, such as studying energy production in mitochondria or protein synthesis in ribosomes.
The technique is also a standard procedure in virology for the purification and concentration of virus particles. Viruses can be separated from host cell components or the culture medium in which they were grown. This isolation is a necessary step for producing vaccines, creating viral vectors for gene therapy, and for basic research into viral structure.
The method is also applied to separate large macromolecules and their complexes. Scientists can isolate protein aggregates, which are implicated in various diseases, or separate specific DNA and RNA molecules from other cellular contents. This ability to fractionate samples makes it a valuable tool in molecular biology, biochemistry, and pharmaceutical development.
Limitations and Complementary Techniques
A primary limitation of differential ultracentrifugation is that it provides a relatively crude separation. The resulting fractions are often not completely pure due to cross-contamination. This happens because smaller, slower-sedimenting particles can become trapped in the pellet along with the larger particles being targeted, leading to a loss of the desired component.
Because of this issue, differential centrifugation is frequently used as an initial purification step rather than a final one. It separates components based on major differences in size, creating enriched fractions that can then be further purified. This makes it a preparatory stage for more advanced separation techniques.
To achieve a higher degree of purity, researchers often use complementary methods like density gradient centrifugation. This technique separates particles within a layered medium, such as a sucrose gradient, based on their density. Methods like rate-zonal and isopycnic centrifugation can separate particles with very similar sizes but different densities, yielding much purer samples.