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 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.
Copyright © 2023 MyJoVE Corporation. All rights reserved