Immunoaffinity Purification of Organelles
The vast majority of subcellular fractionation techniques exploit physical differences between organelles, that is, size, density, charge or hydrophobicity. The most widely used methods are density-gradient centrifugation, free-flow electrophoresis, and polymer-phase partitioning. These approaches are of limited use when organelles with similar physical properties are to be fractionated. In such situations, procedures based on biological differences are required. Of these, methods based on immunological techniques have probably the greatest potential since they rely solely on the presence of specific antigens on the organelles of interest. In 1975, de Duve postulated that each biochemical marker is restricted to a single subcellular site (e. g., cytochrome oxidase in mitochondria). This remains the basis on which the purity of many isolated organelles is assessed, although it has become apparent that subcellular organelles previously thought to be homogeneous may differ in their composition (Reijnierse et al., 1975), while formerly “specific” markers may be found in a variety of subcellular compartments, an example being 5′-nucleotidase (Stanley et al., 1982). Consequently, organelles are now frequently identified by their function (e. g., transport vesicle, synaptic vesicle, transcytotic carrier vesicle) as much as by their composition and appearance under electron microscopes. Since the function of organelles will be reflected in their composition, it is now generally accepted that each functionally distinct organelle has a distinct antigenic pattern. Immunological techniques ought therefore to be capable of isolating any defined organelle, with the proviso that a specific antigen is expressed on the organelle’s surface.
KeywordsCellulose Adenosine Fractionation Polyacrylamide Polystyrene
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