Subcellular analysis cellular fractionation

In general, the aim of subcellular analysis is to quantify an analyte within a specific subcellular compartment. Consequently, in most cases, an organelle fraction must be purified before analysis. Cellular fractionation, that is, isolation and purification of organelles, has been indispensable in the biochemical fields and, as evidenced in the literature, has been used pervasively. Since complete reviews can be found in the biochemical literature, we will only briefly describe the principles of cellular fractionation. 

Enriched subcellular compartments can be analyzed by MS/MS to determine their constituent proteins. One advantage of analysis of different cellular fractions is pre-analytical simplification that offers rewarding yields in dealing with the proteins identified in large-scale MS experiments. One of the major initiatives of the Human Proteome Organization (HUPO) is the comprehensive characterization of the complete subproteome of each cell type. 

In spite of the reduction in complexity that can be achieved by cellular fractionation, an analytical separation is frequently required to separate one or more components from the cellular milieu. As evidenced throughout this book, capillary electrophoresis (CE) provides high resolution and separation efficiency, both of which are necessary for subcellular analysis. In addition, CE is advantageous because it requires very little sample volume, typically less than a nanoliter, and generally very little sample preparation. Hence, capillaries have been used to directly sample subcellular compartments within neurons, oocytes, and muscle tissue sections. They have also been used to analyze individual organelles from a single cell following on-column lysis." ... 

Proteomics research has benefited greatly from subcellular fractionation, because reducing the complexity of the entire proteome to smaller organelle proteomes makes it possible to separate and detect low abundance proteins. Furthermore, since the goal of proteomics is not only to learn protein sequences but also the locahzation and function of proteins, subcellular analysis is advantageous because it provides the subcellular localization. Indeed, the benefits of cellular fractionation before... 

Subcellular fractionation, sucrose density gradients Allows isolation of specific cellular organelles for organelle proteome analysis difficult to obtain completely pure preparations... 

It is well established that in response to a variety of agents that cause an increase in intracellular calcium levels and stimulate LT synthesis, including calcium ionophore, cross-linked IgE receptors, thapsigargin and f-MetLeuPhe, 5-LOX translocates from a soluble to a membrane fraction in cells [26,27,39-41]. By subcellular fractionation and immunoblot analysis, compounds which bind to FLAP both inhibit and reverse this membrane association in a concentration-dependent manner [42—45]. For structural analogues of MK-886 and six different series of quinoline compounds, there is a correlation between inhibition of 5-LOX translocation and cellular LT synthesis [43,46-48], suggesting that FLAP regulates the membrane association of 5-LOX. 

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