Expression systems labeling methods

Fig. 12.3. Some expression systems involving noncovalent (binding) linkages. Numbers correspond to entries (methods) in Table 12.2 (5) labeling with methotrexate derivatives that recognize a dihydrofolatereductase segment, and (11) labeling with biarsenical derivatives binding to a 6-12 tetracysteine...

Although alternative expression systems have been successfully adapted for the production of isotope-labeled proteins (see Sect. 1.5), heterologous expression in E. coli often remains the method of choice for NMR sample preparation. There is a fundamental difference, however, with respect to the kind of medium in which the cells are cultivated. In a so-called chemically defined or minimal medium only one or a very limited number of carbon sources is provided, e. g. glucose or glycerol. All bacterial metabolites have to be biosynthesized by the cells through the various, sometimes lengthy and energy-de-... 

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

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