Subcellular Techniques

This subdivision is not based on the more usual macroscopic criteria it was made possible when techniques of subcellular biology became sufficiently refined for many more fundamental differences to become apparent. Some of the criteria differentiating eukaryotes and prokaryotes are given in Table 1.1. 

Kottke, Electron energy loss spectroscopy and imaging techniques for subcellular localization of elements in mycorrhizas. Methods Mierohiol. 23 369 (1991). 

The calculation of protein proximity and hence association on the basis of sensitized emission or FSPIM requires correction for direct acceptor excitation and donor bleed through using several mathematical models and instrument correction factors [22, 59-61], which is difficult to control [22] (see also Chapters 7 and 8). A high detected acceptor to donor signal ratio in these techniques may also reflect other phenomena than FRET. For instance, this ratio is dependent on cellular expression levels and subcellular localizations, which are difficult to control. Additionally, for the widely used... 

Microanalytical techniques were first pioneered in the 1960s, and the earliest paper using X-ray microanalysis on plant materials is that of Lauchli and Schwander in 1966 (1). It was soon realized that microanalysis could provide a link between anatomical studies and plant physiology. It allowed scientists who were interested in aspects of plant mineral relations to pursue their interests at a cellular or even subcellular level. Microanalysis, in its various forms, is now a well-established technique, and one that is continuing to develop. 

While our data using this technique are still preliminary, we have observed that 25 yU/ml insulin inhibits the rate of calcium efflux from renal slices (28). This effect of insulin was gradually reduced at the higher concentrations of insulin. The effects of insulin on calcium exchange appear to be localized in the mitochondrial compartment. Further work is needed to determine whether insulin affects specific enzyme systems which are known to play a role in renal calcium transport, and which cellular or subcellular compartments are involved. This would substantially increase our understanding of the regulation of urinary calcium excretion, and of ways in which excessive loss of calcium by this route might be avoided. 

There have been rather few studies of the location of probes in whole cells. DPH incorporates into most subcellular fractions (see, e.g, Ref. 64), whereas with TMA-DPH, early after introduction only the plasma membranes appear to be labeled/64,65) There is considerable interest in examining the lipid motional properties of living cells by fluorescence techniques. In this type of study the location of the probe has to be carefully checked before conclusions can be drawn. This is carried out by separate measurements of the recovery of probe from intact labeled cells in isolated subcellular fractions and/or by fluorescence microscopy. 

An extensive variety of in vitro test systems are available that can be used in supplementary investigational studies of the reproductive system. Examples of these systems include the maintenance of whole organs (e.g., isolated perfused testis/ovary), primary culture of gonadal cells and subcellular fractions of organs and cells. In vitro fertilization techniques have also started to show promise in evaluating functional end-points for toxic effects. The information obtained from such test systems can be invaluable in identifying potential mechanisms of action... 

One of the more advanced of the FFF techniques is sedimentation FFF (SdFFF), in which the applied field is a centrifugal force (see Fig. 2.1b). A typical separation achieved through SdFFF is also illustrated in Figure 2.1b. The SdFFF is suitable for species with molecular weights larger than about 106 and has proved useful for a large number of biocolloids (e.g., subcellular particles), polymers, emulsions, and natural and industrial colloids (Giddings 1991). 

Briefly, subcellular fractionation of the tissue is earned out to obtain the fraction containing the protein of interest. This is then subjected to separation in the first dimension by IEF using O FarreH s technique (1), or as described in Methods in Molecular Biology, Volume 3, Chapter 19. At this stage, it is important to obtain complete solubilization of the protein (see Note 1). 

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