Question

How is it that cell fractionation can be used to separate nuclei, lipids, mitochondria, and ribosomes from one another? What properties of the components is/are being used for separation? What can you modify or change about the centrifugation process in order to separate mixtures of components whose properties are very similar?

Answer 1

Cell fractionation procedure for rupturing cells, separation and suspension of cell constituents in isotonic medium from which cell can be separated into its functioning organelles and macromolecules.Cell fractionation 3 steps Extraction, Homogenization and Centrifugation,Organelles Can Be Separated by Ultracentrifugation.

Cells can be broken up from osmotic shock or ultrasonic vibration ground up in a blender. These break many of the membranes of the cell into fragments organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes each with a distinctive size, charge, and density. Provided that the homogenization medium has been carefully chosen the various components derived from the endoplasmic reticulum, called microsomes. cells or tissues are suspended in a solution of appropriate pH and salt content, usually isotonic sucrose (0.25 mol/L) at0-40°C.microsomal fraction requires an ultracentrifugation at 100 000 g to separate the cytosolic soluble fraction from the microsomes.

Centrifugation can then be used to separate the various components of the cellular homogenate, based on their particular size, mass. A low-speed spin will pellet unbroken cells and nuclei; centrifugation of the supernatant from the low-speed spin at a higher will bring down organelles such as the mitochondria; centrifugation of the supernatant from the intermediate speed pellet at yet greater speeds and even longer durations will pellet microsomes (ER) and other small vesicles procedure is termed differential centrifugation. the low-speed nuclear fraction contains not only unbroken cells but also large sheets of plasma membrane wrapped around the nuclei; mitochondrial pellets contain lysosomes and peroxisomes.