Fractionation before Radioactive Labelling

The inactive sample is separated and the fractions obtained are treated with a radioactive reagent, yielding products which are susceptible to radiometric measurement. 

Heavy metal ions, for example, can be separated, treated on the layer with and identified and quantitatively determined as radio- 

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In these types of isotopic analysis, the same compound as that to be measured is used (element or molecule) where one of the atoms in it has been replaced by a radioisotope to allow radioactivity measurements. A small, precisely known quantity of the labelled compound, called the tracer, is added to the sample and, after homogenisation, an aliquot of the spiked sample is isolated by a fractionation technique such as recrystallisation or chromatography. The specific activity of the tracer is measured before and after fractionation. 

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

The preceding method is known as reversed isotope dilution analysis when the compound to be measured is already radioactive. The principle remains the same the activity of the subject compound (measured from a fraction), is carried out before and after dilution with the same compound, non-labelled. The calculations are identical. This analysis is used for the determination of the isotopic carrier in a solution of a radionuclide using one of its stable isotopes. 

Radiochemical purity. The radiochemical purity is the fraction of the stated radioactive nuclide present in the stated chemical form. For tracers of elements stabilized in two or more oxidation states, it is necessary to check their oxidation state by their chemical behavior, ion exchange for example, preferably just before the experiment. In organic compounds labeled with a radioactive nuclide, it is desirable that the number and position of labeling of the radioactive nuclide are unique. However, when the number and position of the nuclide in a compound do not essentially affect its chemical behavior as is often the case in tritium-labeled ones, use of a mixture of a compound labeled with different number of the stated nuclide or labeled at different positions is acceptable. The purity of some labeled compounds decreases gradually due to oxidation, self- or radiolytic decomposition during long storage. Such a labeled compound should be assayed and purified, if necessary, before use. 

Positive identification of receptors in preparations of subcellular fractions is feasible only on conditions that the receptor protein has been labeled before the disruption of the membrane. The labeling techniques commonly employed for the identification of receptor proteins are inadequate, however, since the latter constitute only a minor component of the total cell proteins. In principle, affinity labeling provides the high specificity needed for this purpose. By this method, the reagent interacts reversibly and specifically with the active site of the receptor and then forms a stable covalent bond with amino acid residues located at or near the active site. The attached moiety can be detected by virtue of a tailored chromophore that is radioactive, fluorescent, or spin label. In this connection it is of interest that affinity labeling of the norepinephrine a-receptor was reported as early as 1945. ... 

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