Cell fractionation
Cell fractionation is the process used to separate cellular components while preserving individual functions of each component.[1] This is a method that was originally used to demonstrate the cellular location of various biochemical processes. Other uses of subcellular fractionation is to provide an enriched source of a protein for further purification, and facilitate the diagnosis of various disease states.[2]
Homogenization
Tissue is typically homogenized in a buffer solution that is isotonic to stop osmotic damage. Mechanisms for homogenization include grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, and ultra-sound. The samples are then kept cold to prevent enzymatic damage. It is the formation of homogenous mass of cells (cell homogenate or cell suspension). It involves grinding of cells in a suitable medium in the presence of certain enzymes with correct pH, ionic composition, and temperature. For example, pectinase which digests middle lamella among plant cells.
Filtration
This step may not be necessary depending on the source of the cells. Animal tissue however is likely to yield connective tissue which must be removed. Commonly, filtration is achieved either by pouring through gauze or with a suction filter and the relevant grade ceramic filter.
Purification
Purification is achieved by differential centrifugation – the sequential increase in gravitational force results in the sequential separation of organelles according to their density.
References
- Alberts, B; Johnson, A. "Fractionation of Cells". Molecular Biology of the Cell. 4th edition.
- Ninfa, Alexander J. (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. United States of America: John Wiley & Sons, INC. p. 209. ISBN 978-0-470-08766-4.