Subcellular sampling, with

With the help of a micro-Raman setup the laser spot can be focused down to about 1 pm in diameter. This allows for the differentiation of single bacterial cells or a biochemical analysis of subcellular components within bacterial (diameter approx. 1 pm) or yeast cells (diameter approx. 5-10pm). A confocal Raman setup achieves an even better spatial resolution [6, 7]. This possibility enables Raman mapping or imaging experiments with spatially resolved information of the whole sample in axial and lateral directions. 

Studies with various subcellular fractions are useful to ascertain which enzyme systems are involved in the metabolism of a chug candidate. In the absence of added cofactors, oxidative reactions such as oxidative deamination that are supported by mitochondria or by Ever microsomes contaminated with mitochondria membranes (as is the case with microsomes prepared from frozen liver samples) are likely catalyzed by monoamine oxidase (MAO), whereas oxidative reactions supported by cytosol are likely catalyzed by aldehyde oxidase and/or xanthine oxidase (a possible role for these enzymes in the metabolism of... 

In the late nineteenth century, as physics progressed rapidly, J. J. Thomson discovered the electron the invention of the electron microscope followed several decades later. Because the wavelength of the electron is shorter than the wavelength of visible light, much smaller objects can be resolved if they are illuminated with electrons. Electron microscopy has a number of practical difficulties, not least of which is the tendency of the electron beam to fry the sample. But ways were found to get around the problems, and after World War II electron microscopy came into its own. New subcellular structures were discovered Holes were seen in the nucleus, and double membranes detected around mitochondria (a cell s power plants). The same cell that looked so simple under a light microscope now looked much different. The same wonder that the early light microscopists felt when they saw the detailed structure of insects was again felt by twentieth-century scientists when they saw the complexities of the cell. 

Fluorescently labelled antibodies can be used to visualise cellular or subcellular structures. This is done by incubating antibodies against specific cellular antigens with frozen or fixed tissues sections, or even permeabilised cells (Javois 1994). Unbound antibodies are removed by washing, and then a second anti-immunoglobulin antibody coupled to a fluorescent group, such as fluorescein or rhodamine, is added to the preparation. The sample is washed free of excess fluorescent antibody and visualised using a fluorescence microscope. 

Compared to yeast with about 5800 ORFs which code for proteins, samples from higher organisms can be much more complex and the separation scheme has to be adjusted accordingly. For very simple species as the other extreme, already subcellular fractionation can provide the appropriate pre-separation. For example only one LC-MALDI MS/MS run of a peptide mixture derived from the separated E-coli 50S ribosomal subunit allowed to identify 30 of the 33 expected proteins (Mirgorodskaya et al. 2005a). 

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