Organelles, isolation

The strategy employed is to fractionate cells into organelles and examine the proteins in each organelle. This approach provides smaller mixtures of proteins to be examined, with the prospect of analyzing more proteins overall, allows observations to be correlated within the frame of reference of organelle biology, and occasionally allows the contribution of new observations about which proteins occur in which part of the cell, that is, annotation of the human genome. 

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Adams, R.J.. Pollard, T.D. (1986). Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-1. Nature 322, 754-756. 

Organelle isolation from plant cells is an important technique for the investigation of the physiological functions of the organelle itself. Due to... 

Danley, J.M., Staggers, S., Walker, A., Llewellyn, G.C., and Dashek, W.V. (1981). Aflatoxin-induced alteration in the levels of membrane chemicals of "Essex roots of subcellular organelles isolated from excised, incubated Glycine max, cv. "Essex roots.l.non-enriched organelles. Mycopathologia, 74, 149-161. 

The mechani.sm of photopho.sphorylation i.s chemio.smotic. In 1966, Jagendorf and Uribe experimentally demon.strated for the first time drat establishment of an electrochemical gradient across the membrane of an energy-tran.sdncing organelle conld lead to ATP. synthe.sis. They equilibrated isolated chloroplasts for 60 seconds in a pH 4 bath, adjusted die pH to 8 in die presence of ADP and Pi, and allowed phosphorylation to proceed for 15 seconds. The entire experiment was carried ont in tire dark. 

To correlate embryonic arrests with the metabolic pathways, and especially to understand why cellular organelles first undergo chemical damages, biological investigations include evaluation of DNA, RNA, protein, glucose, lipid, and adenosine-5 -triphosphate (ATP) contents, whose fractions are extracted and isolated by modified Schneider methods. In particular,... 

CTC, used extensively to monitor calcium release in both whole cells and isolated organelles (28-33), is an amphipathic molecule that easily passes through cell membranes (see Figure 1). The fluorescence of this probe is enhanced more than fiftyfold by binding of calcium when the dye is intercalated into biological membranes. 

Wagner, G.J. (1983). Higher plant vacuoles and tonoplasts. In Isolation of Membrane and Organelles from Plant Cells, ed. J.L. Hall and A.L. Moore, pp. 83-118. London Academic Press. 

Because of the difficulties in abundance and compatibility described above, fractionation steps are often performed on protein mixtures prior to 2D gel separation to reduce the complexity of the mixtures. Prefractionation of proteins can be achieved by (i) isolation of cell compartments such as the plasma membrane or organelles such as mitochondria or nuclei, (ii) by... 

Most receptors (a notable exception being the steroid receptors that influence DNA transcription) are located on the cell surface, and purified cell membranes are thus an obvious choice of preparation. When a tissue is homogenized, however, any membrane fraction isolated may well contain membranes from intracellular organelles in addition to cell membranes from all the cell types present in the tissue. Thus, brain membranes will contain membranes not only from neurons but... 

The biocatalyst is incorporated in different physical forms as an isolated enzyme, a whole cell, a subcellular organelle or a tissue slice depending on... 

Video microscopy allows study of molecular mechanisms through direct observation of organelle movements while precise control of experimental conditions is maintained. Fast axonal transport continues unabated in isolated axoplasm from giant axons of the squid Loligo pealeii for hours [14]. Video microscopy applied to isolated axoplasm permits a more rigorous dissection of the molecular mechanisms for fast axonal transport... 

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