Culture-Based Labeling Methods

A different approach to the same general objective was first suggested (Veenstra 2000) in a somewhat different context, that of assisting interpretation of LC-MS data for intact proteins (no digestion involved) to identify the proteins using a combination of accurate molecular 

Finally in this section, it must be emphasized that relative quantitative proteomics measurements currently use a range of methodologies other than those based on mass spectrometry. Recently (Unwin 2006) this range of techniques, including those summarized above plus protein arrays and flow cytometry, has been reviewed 

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Latter et al have recently published the results of a study in which cells growing in culture were labelled with 20 radioactive amino acids, each in a different experiment, and analyzed by 2D6EL (S4). The resulting autoradiographs were studied with an image analysis system so that amino acid compositional data could be extracted. This information was then compared to the amino acid composition data resident in two large data bases, using computerized search methods. Out of the 122 spots studied in this manner, tentative identifications could be made on 17 of them based on the amino acid composition alone. 

Microarray detection methods In the early days of protein microarray analysis, mostly radioisotope-based labeling was applied, especially for phosphorylated proteins [99]. Later, stable isotope-coded amino acids (SILAC) proved useful in in vivo incorporation to cell cultures [100]. Both methods were very sensitive, requiring only minute sample amounts to reveal protein expression differences [101]. The use of an enhanced chemiluminescence (ECL) technique using X-ray film exposure or a phosphor imager instrument was. 

The efficacy of some modifications in correcting the assay were studied. Skin biopsies were removed and cultured by standard methods (3). Cell extracts were prepared by freezigg and thawing of concentrated cell suspensions (>6x10 /ml) in the presence of 1 mM PRPP. HGPRT and APRT were determined in the dialyzed supernatants of the cell extracts by a radiochemical method in which the labelled purine bases are converted to their respective nucleotides by reacting with PRPP,... 

Most biochemical methods are based on the assimilation of CO2 by plants and the feeding of various kinds of microorganisms or animals with labelled compounds. Afterwards, the compounds synthesized in the plants, microorganisms or animals are isolated. In this way, glucose, amino acids, adenosine triphosphate, proteins, alkaloids, antibiotics, vitamins and hormones can be obtained that are labelled with or Cultures of various microorganisms such as clorella vulgaris may be applied and operated as radionuclide farms . The labelled compounds produced by biochemical methods are, in general, labelled at random, i.e. not at special positions. 

Systemic bioavailability is the product of fraction of dose absorbed (/a), fraction of dose escaping gut metabolism (/g), and fraction of dose escaping first-pass metabolism (F ). Permeability class is based upon /a, which may be estimated either in vivo or in vitro by direct measurement of mass transfer across human intestinal epithelium. In vivo methods include (i) mass balance studies using unlabeled, stable-isotope labeled, or a radiolabeled drug substance (ii) oral bioavailability using a reference intravenous dose or (iii) intestinal perfusion studies either in humans or an acceptable animal model. Suitable in vitro methods involve the use of either excised human/animal intestinal tissues or cultured epithelial monolayers. All of these methods are deemed appropriate for drugs whose absorption is controlled by passive mechanisms. 

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