Labelling biochemical methods

R F Schall and H J Tenoso, Alternatives to Radioimmunoassay Labels and Methods, C// Chem, 1981,27,1157-1164 D S Smith et al, A Review of Fluoroimmunoassay using Immunofluorometnc Assay, nn dm Biochem, 1981,28, 253-274... 

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. 

Biochemical Assays. In vitro biochemical methods are based on the fact that TSH will release from thyroids previously labeled with by incubation of guinea pig thyroid slices with the labeled iodine (Bll). El Kabir (El) improved the method by treating the guinea pigs with a goitrogen prior to removal of the thyroids. 

In another approach, the colony hybridization technique (Figure 18D, bacteria are screened by using a radioactively labeled nucleic acid probe, an RNA molecule or a single-stranded DNA molecule with a sequence complementary to that of a specific sequence within the recombinant DNA. Bacterial cells are plated out onto solid media in petri dishes and allowed to grow into colonies. Each plate is then blotted with a nitrocellulose filter. (Most of the original colonies remain on the petri dishes.) The cells on the nitrocellulose filter are lysed, and the released DNA is treated so that hybridization with the probe can occur. Once nonhybridized probe molecules have been washed away, autoradiography (Biochemical Methods 2.1) is used to identify the colonies on the master plate that possess the recombinant DNA. 

Yan and his colleagues used a stable isotope labeled proteome internal standard to analyse protein expression alterations during oxidative stress in breast epithelial cells. Affinity chromatography was combined with MS/MS in the proteomic analysis of human 06-methylguanine-DNA methyltransferase. These studies demonstrated that proteomics can elucidate protein expre-ssions associated with the pharmacological and toxic effects of an anticancer drug when integrated with other biochemical methods. 

This property can be exploited with a two-color pulse chase of protein turnover in living cells, using the sequential addition of FlAsH and ReAsH to label old and newly synthesized proteins. The time course of protein turnover can be determined simply by varying the time interval between removal of the first label and the addition of the second label. Unlike traditional biochemical methods of pulse chase that use incorporation of radioactive amino acids, the two-color method allows continuous imaging of single cells and can reveal additional information about subcellular localization of protein turnover. 

C]taurocholic acid (201) and [7-3H]dehydroepiandrosterone sulphate (202) in [2H5]glycerol matrix have been used184 to interpret the partial negative ion FAB spectra of these unlabelled compounds and to show that FAB and field desorption mass spetrometry enable one to determine quickly the specific radioactivity and label distribution within highly labelled biochemicals. The minimal specific radioactivities detectable by this method are about 20MBq/mmol and lOGBq/mmol for compounds labelled with 14C and with tritium, respectively, with 1% and 5% accuracy. The use of mass spectrometry minimizes interferences from labelled impurities. 

A new biochemical method different from the LAL assay is currently under development (Associates of Cape Cod). This is a direct fluorescence method that uses a labeled tracer reactive with endotoxin. The tracer is a recombinant DNA-prepared LPS binding protein. No published studies are yet available that have evaluated this method against the LAL assay. 

The DAG kinase assay is currently the most commonly employed biochemical method for the quantification of total cellular DAG. This assay utilizes recombinant bacterial DAG kinase to phosphorylate DAG that has been extracted from the cell with the method of Bligh and Dyer (1959). Utilization of p P] ATP as a source of phosphate allows the direct detection of P-labeled DAG via autoradiography and scintillation counting. The DAG kinase assay is linear over a range of 25 pmol to 20-25 nmol of DAG, and is very useful for the elucidation of biochemical pathways (Kennerly et al, 1979 Preiss et al, 1987). The only drawback to this approach is its inability to identify individual DAG species. 

As with their unlabeled counterparts, e.p. tritiated or carbon-14-labeled compounds can be prepared in three ways resolution of racemates, stereoselective syntheses and biochemical methods. Each of these methods has its own strengths and limitations, according to the structural complexity of the compound, the availability of nonlabeled reference material(s) and the kinds of in-house experience and resources available. The resolution of racemates and stereoselective syntheses are the topics of this chapter. Biochemical methods are the subject of Chapter 12. 

Chemistry-based kinetic resolution methods, which make use of the preferential reaction of one enantiomer with a chiral reagent (e.g. hydroboration of racemic alkenes with diisopinocampheylborane) or an achiral reagent in the presence of an appropriate chiral catalyst (e.g. Sharpless epoxidation of racemic allylic alcohols with t-BuOOH in the presence of (2R,3R)- or (25,35)-diisopropyl tartrate and Ti(Oi-Pr)4) have not been exploited so far for the isolation of e.p. labeled substances. In contrast, biochemical methods have been widely used, particularly for the resolution of racemic a-[ " C]amino acids and various [ C]carboxylic acids. Such methods, including ... 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Chetrit, P., Gaudin, V., de Courcel, A., and Vedel, F. (1989) A cross-hybridization method for DNA mapping with photobiotin-labeled probes. Anal. Biochem. 178, 273-275. 

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