Preparation of extracts from animal tissues.

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. The subcellular distribution of the protein or enzyme complex should be considered. If located in a specific cellular organelle such as the nuclei, mitochondria, lysosomes, or endoplasmic reticulum, then an initial subcellular fractionation to isolate the specific organelle can lead to a significant degree of purification in the first stages of the experiment (1). Subsequent purification steps may also be simplified, as contaminating proteins may be removed in the centrifugation steps. In addition, the deleterious affects of proteases released as a result of the disruption of lysosomes may also be avoided. Proteins may be released from organelles by treatment with detergents or by disruption resulting from osmotic shock or ultrasonication. Although there is clearly an ad-