Login-Verarbeitung ...

Trial ends in Request Full Access Tell Your Colleague About Jove

32.6: Subcellular Fractionation

JoVE Core
Cell Biology

Ein Abonnement für JoVE ist erforderlich, um diesen Inhalt ansehen zu können. Melden Sie sich an oder starten Sie Ihre kostenlose Testversion.

Subcellular Fractionation

32.6: Subcellular Fractionation

The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular fractionation. These isolates are used to study specific cellular components, analyze localized protein activity, and are even employed in diagnostics. Fractionation is typically achieved using centrifugation methods, the most common being density-gradient and differential centrifugation.

Differential Centrifugation

Differential centrifugation is a relatively simple  method that separates the cellular components based on size and density. Sequential centrifugation with increasing speeds (ranging from 10,000 X g to 150,000 x g) sediments the differently sized components. However, since multiple organelles can be of similar size and density, this method usually produces crude fractions.

Density Gradient Centrifugation:

Highly purified fractions of cellular components can be obtained by separating the homogenate in a density gradient solution. A density gradient is prepared in a centrifuge tube by layering solutions with increasing densities, such as increasingly concentrated sucrose solutions, with the densest layer at the bottom of the tube. Such gradients are used in rate-zonal centrifugation to separate cellular organelles based on their size and shape. Upon centrifugation, the organelles sediment at different rates, based on their sedimentation coefficients, as they move through the different density layers.

Alternatively, a continuous density gradient can also be prepared by mixing solutions of different densities in gradual proportions along the length of the tube. During centrifugation, each component immobilizes at the position that matches their density — their equilibrium position. Hence this method is also known as equilibrium or buoyant sedimentation. This separation of cellular components and molecules is thus based on their density, not their size.

Suggested Reading


Keywords: Subcellular Fractionation Membrane-bound Organelles Density-gradient Centrifugation Differential Centrifugation Sedimentation Density Gradient Rate-zonal Centrifugation Equilibrium Sedimentation Cellular Components Organelle Purification

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter