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.

Subcellular fractionation is used to obtain pure fractions of different cell organelles from a cell lysate.

Two commonly used methods  for subcellular fractionation are  differential centrifugation and density gradient centrifugation.

Differential centrifugation uses sequential centrifugation at progressively increasing speeds for size-based separation of organelles.

First, the sample is centrifuged at a low speed of around 400 to 600 x g to sediment large structures like nuclei and cell debris.

Then, the supernatant is spun at high speed ranging from 10,000 to 20,000 x g to pellet organelles such as mitochondria, lysosomes, and peroxisomes.

Further centrifugation cycles at speeds more than 80,000 x g can separate microsomes, membrane fractions, and even ribosomes.

However, differential centrifugation cannot separate organelles that are very close in size or density, such as mitochondria from peroxisomes.

In such cases, density gradient centrifugation, a more sensitive method, is a valuable tool.

This method  uses density gradients established using chemicals

such as sucrose or glycerol to separate organelles into distinct layers based on size, shape, or density within a single tube.