Differential Centrifugation: Videos & Practice Problems

After extracting proteins, the next crucial step in protein purification is differential centrifugation. Centrifugation is a technique that utilizes a centrifuge, a machine designed to spin samples at high speeds, creating a centrifugal force that separates components based on their density and size. The crude extract obtained from protein extraction contains a mixture of various proteins and other molecules.

During centrifugation, insoluble proteins and particles, which do not dissolve, are pulled down to the bottom of the test tube, forming a solid mass known as a pellet. The liquid remaining above this pellet is referred to as the supernatant, which contains soluble proteins and other unprecipitated solutes. The behavior of these particles during centrifugation is characterized by the sedimentation coefficient, measured in Svedberg units (S). This coefficient indicates how quickly a particle settles in a centrifugal field; a higher S value corresponds to faster sedimentation.

The sedimentation coefficient is influenced by several factors, including the particle's density, shape, and mass, as well as the properties of the solvent. For instance, proteins with a higher molecular weight generally exhibit a larger sedimentation coefficient, but this is not a strict rule. An example is ribonuclease A, which has a greater molecular weight than cytochrome c, yet cytochrome c has a higher sedimentation coefficient. This discrepancy highlights that factors such as density and shape can also significantly affect the sedimentation behavior of proteins.

In practice, after centrifugation, the pellet containing the denser proteins can be separated from the supernatant by carefully pouring off the liquid. This process allows for the isolation of specific proteins based on their sedimentation characteristics, setting the stage for further purification steps, such as differential centrifugation, which will be explored in subsequent lessons.

Differential centrifugation is a crucial technique in protein purification that involves the stepwise separation of cellular components based on their sedimentation coefficients. After obtaining a crude extract through cell homogenization, the process begins by placing this extract into a centrifuge. By spinning the extract at specific velocities for precise durations, different organelles and cell components can be pelleted, allowing for their separation from the supernatant, which contains unpelleted materials.

Initially, when the crude extract is centrifuged, a pellet forms at the bottom of the test tube, containing the nuclei of the cells, while the supernatant remains above. The supernatant can be carefully transferred to a new test tube, leaving the nuclei pellet behind. This process can be repeated with the supernatant, spinning it again at a different velocity to pellet other components, such as mitochondria. Each subsequent centrifugation step allows for the isolation of specific organelles, including ribosomes and insoluble proteins, by adjusting the spinning conditions.

Throughout this process, the focus may shift between the pellet and the supernatant depending on the target component of interest. For example, if the goal is to isolate proteins located within the nuclei, the pellet containing the nuclei can be retained for further analysis. Conversely, if soluble proteins are the target, the supernatant is of primary interest. Although differential centrifugation effectively reduces the complexity of the mixture by removing unwanted components, further purification steps are often necessary to achieve a high level of protein purity.

In summary, differential centrifugation is a systematic approach to separate cellular components based on their physical properties, facilitating the purification of proteins for subsequent analysis and experimentation.