The fast, efficient, and accurate release of proteins from cells and tissues is a critically important initial step in most analytical processes, and is essential to reliable proteomic analyses. Two-dimensional gel electrophoresis (2DGE) can be an accurate representation of a proteome only if the entire protein constituency of cells is recovered during the sample preparation process. The use of chaotropes and new surface-reactive agents has substantially improved the capacity of 2DGE to produce more comprehensive maps of cellular and subcellular proteomes, but this increased stringency frequently complicates downstream analyses such as electrophoresis, chromatography, and mass spectrometry. Further, the disruption of cells and tissues usually requires coupling these chemistries with mechanical disruption methods which may contribute to deleterious effect. Pressure Cycling Technology (PCT) uses alternating cycles of high and low hydrostatic pressure to effectively induce the lysis of cells and tissues in preparation for 2DGE and other analytical or preparative methods. Pulses of high hydrostatic pressure perturb equilibria between cellular components causing them to dissociate without breaking covalent bonds. PCT combined with the appropriate extraction buffers is particularly efficient for the dissolution of membrane proteins and other traditionally difficult analytes. PCT derived 37% more protein from the nematode Caenorhabditis elegans than sonication and 17.1% more protein from the gram-negative bacteria Rhodopseudomonas palustris than enzymatic lysis. Remarkably, PCT enabled 2DGE analysis from a single Daphnia pulex organism where over 500 protein spots were detected in silver stained gels. For mammalian tissues, PCT isolated more protein from liver and adipose, both quantitatively and qualitatively, as demonstrated by the appearance of several unique proteins in 2DGE which were not recovered by conventional homogenization techniques. Improved protein recoveries have also been demonstrated for plant tissues where PCT yielded three times more protein from Strelitzia reginae sepals than a centrifugal homogenizer. Moreover, PCT facilitates precise control over sample processing conditions and ensures greater reproducibility.

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