Preparation of Extracts From Animal Tissues
Part of the
Methods in Molecular Biology
book series (MIMB, volume 244)
The initial procedure in the isolation of an protein, a protein complex, or a subcellular organelle is the preparation of an extract that contains the required component in a soluble form. Indeed, when undertaking a proteomic study, the production of a suitable cellular extract is essential. Further isolation of subcellular fractions depends on the ability to rupture the animal tissues in such a manner that the organelle or macromolecule of interest can be purified in a high yield, free from contaminants and in an active form. The homogenization technique employed should, therefore, stress the cells sufficiently enough to cause the surface plasma membrane to rupture, thus releasing the cytosol; however, it should not cause extensive damage to the subcellular structures, organelles, and membrane vesicles. The extraction of proteins from animal tissues is relatively straightforward, as animal cells are enclosed only by a surface plasma membrane (also referred to as the limiting membrane or cell envelope) that is only weakly held by the cytoskeleton. They are relatively fragile compared to the rigid cell walls of many bacteria and all plants and are thus susceptible to shear forces. Animal tissues can be crudely divided into soft muscle (e.g., liver and kidney) or hard muscle (e.g., skeletal and cardiac). Reasonably gentle mechanical forces such as those produced by liquid shear may disrupt the soft tissues, whereas the hard tissues require strong mechanical shear forces provided by blenders and mincers. The homogenate produced by these disruptive methods is then centrifuged in order to remove the remaining cell debris.
KeywordsAnimal Tissue Integral Membrane Protein Surface Plasma Membrane Rigid Cell Wall Mince Tissue
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Claude, A. (1946) Fractionation of mammalian liver cells by differential centrifugation: II. Experimental procedures and results. J. Exp. Med.
, 61–89.CrossRefGoogle Scholar
Rabilloud, T. (1995) A practical guide to membrane protein purification. Electrophoresis
, 462–471.CrossRefGoogle Scholar
Arigita, C., Jiskoot, W., Graaf, M. R., and Kersten, G. F. A. (2001) Outer membrane protein purification. Methods Mol. Med.
, 61–79.PubMedGoogle Scholar
Smith, A. L. (1967) Preparation, properties and conditions for assay of mitochondria: slaughterhouse material, small scale. Methods Enzymol.
, 81–86.CrossRefGoogle Scholar
Tyler, D. D. and Gonze, J. (1967) The preparation of heart mitochondria from laboratory animals. Methods Enzymol.
, 75–77.CrossRefGoogle Scholar
Dignam, J. D. (1990) Preparation of extracts from higher eukaryotes. Methods Enzymol.
, 194–203.PubMedCrossRefGoogle Scholar
Völkl, A. and Fahimi, H. D. (1985) Isolation and characterization of peroxisomes from the liver of normal untreated rats. Eur. J. Biochem.
, 257–265.PubMedCrossRefGoogle Scholar
Ernster, L. and Nordenbrand, K. (1967) Skeletal muscle mitochondria. Methods Enzymol.
, 86–94.CrossRefGoogle Scholar
Scarpa, A., Vallieres, J., Sloane, B., and Somlyo, A. P. (1979) Smooth muscle mitochondria. Methods Enzymol.
, 60–65.PubMedCrossRefGoogle Scholar
Blobel, G. and Potter, V. R. (1966) Nuclei from rat liver: isolation method that combines purity with high yield. Science
, 1662–1665.PubMedCrossRefGoogle Scholar
Nedergaard, J. and Cannon, B. (1979) Overview—preparation and properties of mitochrondria from different sources Methods Enzymol.
, 3–28.PubMedCrossRefGoogle Scholar
Chappell, J. B. and Perry, S. V. (1954) Biochemical and osmotic properties of skeletal muscle mitochondria. Nature
, 1094–1095.PubMedCrossRefGoogle Scholar
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