Cellular fractionation method

With advances in separation media, organelles can now be separated based on their density, size, or both. This makes centrifugation the most versatile and practical cellular fractionation method. 

Separation into components can only be achieved by stopping the process when sedimentation of the desired component has occurred. The sediment is then resuspended in fresh solvent and centrifuged at a lower speed, when the heavier particles will sediment leaving the component in suspension. Such a method is known as differential sedimentation and is particularly useful for the fractionation of cellular components. The method outlined in Procedure 3.3 is simple and is designed to separate four main cellular fractions, namely, nuclear, mitochondrial, microsomal and soluble. 

Oxidation of other forms of cerebrosides by galactose oxidasel Microsomes (5.49 mg protein) and cytosol (7.15 mg protein) from 25 day-old rat brain, which were prepared as described previously (15), were each incubated with 100 units of galactose oxidase overnight at room temperature. The results indicate that oxidation of cerebrosides in these brain sub-cellular fractions was not detected. We tested whether cerebrosides in artificial membrane could be oxidized by this enzyme. Liposomes were prepared as described in Procedures. 0.2 ml Of the liposomes which contained 40 yg nonhydroxycerebroside and 33 yg hydroxycerebroside was incubated with 100 units of galactose oxidase at room temperature overnight. The examination of the product as described above shows that cerebrosides in liposomes was not oxidized. Another aliquot (0.2 ml) of the liposomes was mixed with 1 ml tetrahydrofuran, 1.0 ml of 10 mM phosphate buffer, pH 7.2 containing 100 units of galactose oxidase. More than 90% of cerebrosides were oxidized by this method. 

Thus, the NMR approach gives a most precise picture of the PolyP content and polymerization degree in different cell compartments by a combination of NMR spectroscopy with the methods of sub-cellular fractionation and chemical extraction of PolyPs. 

Methods for cell and sub-cellular fractionation include immuno-isolation, electromigration e.g. free flow electrophoresis), flow cytometiy, density gradient isolation of organelles and sequential extraction. The use of some of these approaches to investigate the mitochondrial and myofibrillar sub-proteomes of the heart will be described in Sections 3.4 and 3.6 respectively. A particular problem in proteomic analysis of the heart is the diversity of cell types that are present. Proteomic profiles of total myocardial lysates are dominated by the proteins present in cardiac myocytes, but such samples... 

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,... 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Quantitative studies comparing the relative abundances of proteins in different cellular states may be performed with MS. Methods such as 2D-GE have been utilized extensively with great success and differentially represent spots excised and then subjected to MS/MS for final identification of the differentially expressed proteins. 2D-GE requires approximately 50 pg of starting material and is limited by its bias toward high abundance proteins and propensity to detect proteins with extreme pi values. Furthermore, proteins at both extremes of molecular weight and those associated with membrane fractions are not well represented by 2D-GE.10... 

Many methods for the fractionation of cellular organelles have been described and it is probably not possible to have a method that is equally effective at preserving each individual component. For each type of cell and organelles the most appropriate method must be selected and evaluated. 

Numerous testing systems and protocols have been used to study 5-LO inhibitors in different laboratories, complicating attempts to compare compounds and series. In vitro, a variety of both cell-free and cellular preparations have been employed as primary screens. The most commonly used cell-free system is the crude cytosolic fraction from broken RBL-1 cells [25] various broken neutrophil preparations are also used, and more recently purified enzymes have occasionally been employed. The formation of 5-LO products is generally determined by radioimmunoassay or (in older work) HPLC or bioassay methods. 

Rather, the isolated subcellular fraction is a mitochondrial fraction, which indicates that its major components are mitochondria. Other cellular components that may be present are lysosomes, cell fragments, nuclear fragments, and microbodies (peroxisomes). The purity of the fraction depends on the source of the extract and the method chosen for isolation. 

The q-space imaging method, which deals with signals only after long diffusion times, discards all information relevant to dynamic aspects of water diffusion and transport, especially the restriction of water transport by membrane and cell wall permeability barriers in cellular tissues. This information is contained in the functional dependence of the pulsed gradient spin echo amplitude S(q,A,x) on the three independent variables q, A, and x (x is the 90-180 degree pulse spacing) [53]. As the tool to explore the q and A dependence of S(q,A,x), generalized diffusion times and their associated fractional populations are introduced and a multiple exponential time series expansion is used to analyze the dependence [53]. 

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