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Posted on Thu, May 10, 2018

Differential Centrifugation in bio separation

The force of gravity will cause sedimentation of particles from a heterogeneous mixture; larger and denser particles sedimentfaster than the smaller and less dense particles. This phenomenon is useful for separating heterogeneous solutions into independent components, and for the isolation and enrichment of target molecules, cells, and cell organelles. Differential centrifugation accelerates the separation process by introducing centripetal forces many times greater than gravity. The precipitated particles form a pellet at the bottom of the tube during centrifugation. The rate of sedimentation is dependent on the size and density of the particles, so centrifugation can be used to isolate target particles simply by controlling centrifugal force or the rate of centrifugation. The rate of centrifugation is reported as angular velocity by revolutions per minute (rpm) or as acceleration(g). RPM is dependent on the radius of the rotor in the centrifuge.

 

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Differential centrifugation vs density gradient centrifugation

There are two main methods of separating particles by centrifugation: differential centrifugation and density gradient centrifugation. Differential centrifugation capitalizes upon the differential rates of sedimentation of particles of varying density. The larger, denser particles have a higher rate of sedimentation. Density gradient centrifugation produces a cleaner separation of particles than differential centrifugation by employing a density matrix for the particles to move through.

The differential centrifugation process involves multiple centrifugation steps of incrementally increased centrifugal force. The largest and densest particles with the greatest rate of sedimentation will comprise the pellet during the initial low-force spin while the smaller, less dense particles remain in the supernatant. The pellet and supernatant can then be separated, and the supernatant can be placed back into the centrifuge at a higher centrifugal force to pull out the next group of particles with a lower rate of sedimentation. This process is repeated as many times as necessary to isolate each desired group of particles. As an example, cells that are lysed in a detergent-free buffer and all membrane-bound proteins will remain associated with their specific membranes. After centrifugation the soluble cytosolic proteins will be located in the supernatant while the membrane associated proteins will be in the heavier pellet. To further enrich proteins from a specific cellular organelle, differential centrifugation can be used. Nuclei will form a pellet when centrifuged at 600 g for 10 minutes. The supernatant can be further centrifuged at 15,000 g for 10 minutes to bring mitochondria and lysosomes into the pellet, while the supernatant from this step can be centrifuged again at 100,000 g for 10 minutes to collect a microsomal pellet. The heterogeneity of biological particles makes isolates from differential centrifugation prone to contamination and poor recovery. This issue can be fixed by washing the pellet and repeating the centrifugation protocol, and by further filtering the sample.

Density gradient centrifugation employs a tube packed with a material that forms a gradient of increasing density and viscosity. Various types of media are used for density gradient separation including polyhydric alcohols, polysaccharides, inorganic salts, and silica. The type of matrix used is chosen based on the target molecule. The density gradient matrix allows for more stringent separation of particles with less contamination. The particles move through the matrix at different sedimentation rates and settle out into clean bands. 

 

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Biomagnetic separation and centrifugation

Centrifugation and filtration are both important steps in bio separation protocols. Biomagnetic separation can speed up the process by quickly binding and isolating targets from a heterogeneous solution (positive selection) or by binding and washing out contaminants (negative selection). A combination of differential centrifugation and biomagnetic separation is used in many isolation protocols to achieve a highly enriched target population. 

 

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