Name: separating cell organelles by centrifugation
Uploaded: 2023-04-30T11:57:44.000Z
Duration: 1 min 32 s
Channel: JoVE

Subcellular fractionation is used to obtain pure fractions of different cell organelles from a cell lysate.
Two commonly used methods&#160; for subcellular fractionation are&#160; 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&#160; 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 tub

subcellular fractionation

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&#160; 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 &#8212; 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.

The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular ...

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Cell Biology

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Analyzing Cells and Proteins

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separating cell organelles by centrifugation

Separating cell organelles by centrifugation lets scientists isolate membrane-bound parts of a cell after lysis. The starting material is a homogenate, which is the mixture left after cells are broken open. These isolated fractions help researchers study specific cell parts, measure protein activity in one location, and support some diagnostic tests.

Fractionation usually uses centrifugation, which spins the sample to separate its contents. The two main methods are differential centrifugation and density-gradient centrifugation. Differential centrifugation is the simpler method. It separates cellular components mainly by size and density.

In differential centrifugation, the sample is spun several times at increasing speeds, often from 10,000 x g to 150,000 x g. Larger or denser components sediment first, and smaller ones remain in the liquid until a faster spin. Because different organelles can be close in size and density, this method usually gives crude fractions rather than highly pure ones.

Density-gradient centrifugation gives more purified fractions. It uses a density gradient solution in a centrifuge tube. The tube is layered with solutions of increasing density, such as sucrose solutions that become more concentrated toward the bottom. These gradients can be used in rate-zonal centrifugation, where organelles separate based on size and shape as they move through the layers at different speeds.

A continuous density gradient can also be made by mixing solutions so the density changes gradually along the tube. During centrifugation, each component stops at the position that matches its density. This is called equilibrium sedimentation or buoyant sedimentation. In this method, separation depends on density, not on size.

Separating Cell Organelles by Centrifugation

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Separating cell organelles by centrifugation lets scientists isolate membrane-bound parts of a cell after lysis. The starting material is a homogenate, which is the mixture left after cells are broken open. These isolated fractions help researchers study specific cell parts, measure protein activity in one location, and support some diagnostic tests. Fractionation usually uses centrifugation, which spins the sample to separate its contents. The two main methods are differential centrifugation...

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key terms and concepts

Learn how differential and density-gradient centrifugation separate cell organelles after lysis, and why these methods are used to study cellular fractions.

Separating Cell Organelles by Centrifugation | JoVE K12

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Separating Cell Organelles by Centrifugation

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