Subcellular Fractionation

Subcellular fractionation obtains pure fractions of different cell organelles from a cell lysate. These isolates are used to study specific cellular components, analyze localized protein activity, and are employed in diagnostics. The process follows tissue homogenization and cell lysis to separate membrane-bound organelles into distinct, usable fractions for research and analysis.

Differential centrifugation uses sequential centrifugation at progressively increasing speeds for size-based separation. The sample is first centrifuged at low speed around 400-600 x g to sediment nuclei and cell debris, then at higher speeds up to 20,000 x g to pellet mitochondria, lysosomes, and peroxisomes. Further cycles at speeds exceeding 80,000 x g separate microsomes, membrane fractions, and ribosomes.

Differential centrifugation cannot separate organelles that are very close in size or density, such as mitochondria from peroxisomes. This method typically produces crude fractions rather than highly purified isolates. When organelles share similar physical properties, more sensitive separation techniques are required to achieve pure fractions.

Density gradient centrifugation is a more sensitive method that uses density gradients established with chemicals like sucrose or glycerol. Organelles separate into distinct layers based on size, shape, or density within a single tube. This approach produces highly purified fractions of cellular components compared to differential centrifugation.

Rate-zonal centrifugation separates organelles based on their sedimentation coefficients as they move through different density layers at different rates. Equilibrium centrifugation, also called buoyant sedimentation, allows each component to immobilize at its equilibrium position matching its density. Equilibrium separation depends on density, while rate-zonal separation depends on both size and sedimentation rate.

Density gradients are prepared using chemicals such as sucrose or glycerol. These solutions are layered with increasing densities, with the densest layer at the bottom of the tube. A continuous density gradient can also be prepared by mixing solutions of different densities in gradual proportions along the tube length.

Subcellular fractionation enables researchers to isolate and study specific cellular components and analyze localized protein activity within organelles. By obtaining pure fractions of different organelles, scientists can determine which proteins are present in specific cellular compartments and understand their functional roles in distinct cellular environments.