Labeling procedures

Generally, methylation of enolate ions with isotopically tagged methyl iodide is a satisfactory labeling procedure. For example, application of this method has given the C-18 labeled steroids, (244) and (245) (see above), 17 -acetoxy-4jS-trideuteriomethyl-4a-methyl-l9-norandrost-5-en-3-one (264) and 19- C-testosterone acetate (268). Methylation of the anion derived from 17jS-acetoxy-4-methyl-l9-norandrost-4-en-3-one (263) with d3-methyl iodide occurs predominantly at C-4, yielding mainly the 4)S-trideuterio-methyl derivative (264) and about 10% of the corresponding C-4 epimer... 

Plants grown for longer periods in solid supports such as sand or soil repre-.sent the next level of complexity and, although other techniques are available, carbon flow is most frequently estimated using C labeling experiments. In the laboratory, COt can be supplied to shoots either as a short pulse or continuously, and the carbon flow can be monitored. In the field, due to technical limitations, only COi pulse labeling procedures are possible. A final approach, termed crop studies, involves the measurement of components of crop growth from which... 

Preparation of Reagent and Labelling Procedures. The structure of F-D [2-(2,4-diazobicyclo-2,2,2-octyl)-4-(5-aminofluoresceinyl)-6-morpholinyl 1,3,5-triazine] has been confirmed by its FAB-MS, IR, and H-NMR spectra (9). Briefly, F-D was synthesized by the treatment of fluorescamine isomer I with cyanuric chloride, then reaction with morpholine and DABCO (l,4-diazobicyclo-2,2,2-octane), as illustrated... 

The results obtained with the ICAT labeling strategy are similar, in principle, to those obtained by in vivo labeling with stable isotopes in that the relative ratios of proteins from different samples are obtained (Fig. 3.2). The important difference between the methods is that the ICAT-labeling procedure is performed in vitro on protein lysates. Therefore, the ICAT-labeling strategy can be applied to samples that cannot be labeled in vivo. Because the ICAT-labeling method can be used with virtually any sample, it... 

Aghajanian, G. K. Vandermaelen, C. P. (1982). Intracellular identification of central noradrenergic and serotonergic neurons by a new double labeling procedure. J. Neurosci 2, 1786-92. 

In some cases, the preparation of a fluorescently labeled antibody is not even necessary. Particularly, if indirect methods are used to detect antibody binding to antigen, then preparing a fluorescently labeled primary antibody is not needed. Instead, the selection from a commercial source of a labeled secondary antibody having specificity for the species and class of primary antibody to be used is all that is required. However, if the primary antibody needs to be labeled and it is not manufactured commercially, then a custom labeling procedure will have to be done. 

Regardless of the type of enzymatic labeling used, it is important that the label be incorporated into the nucleoside triphosphates or primers in a way that does not affect enzyme recognition and activity. Thus, every enzymatic labeling procedure for modifying RNA or DNA probes must start with chemical derivatization of individual nucleotides. Of the many chemical procedures that can be used to modify a nucleoside triphosphate monomer, there are only a few that will result in a derivative still able to be enzymatically added to an existing oligonucleotide strand. 

Ease of separation of tritiated products from a reaction medium is an important feature in the choice of labeling procedure. Sometime ago we used polymer-sup-ported acid and base catalysts [12, 13] to good effect and with the current interest in Green Chemistry one can expect to see more studies where the rate accelerations observed under microwave-enhanced conditions are combined with the use of solid catalysts such as Nafion, or zeolites. 

The most widely applied principle is haptenylation of amino groups via A-hydroxy succinimide esters (NHS-ES). For convenient protein-labeling procedures,... 

Subsequent to incidents involving peroxidation of stored bottles of vinylidene chloride, a labelling procedure and list of peroxidisable compounds was prepared [3], Of the 108 compounds listed, 35 are noted as forming peroxides with ease, and these need particular care in storage and use. A revised data sheet is now available [4], and peroxide-containing residues may often be rendered innocuous by pouring into an excess of sodium carbonate solution [5],... 

E. P. Diamandis and R. C. Morton, Time-resolved fluorescence using a europium chelate of 4,7-bis(chlorosulfophenyl)-l,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Labelling procedures and applications in immunoassays, J. Immunol. Methods 112, 43-52 (1988). 

Once the dissociative mechanism is established, it is possible to apply Gutmann s theoretical treatment (40) to the elucidation of the rate-determining step of the exchange reaction. For deuterium-tritium double labeling procedures, i.e., D O 100%, TgO 1%, it may be shown that the following normalized equations apply under initial exchange conditions ... 

Another fluorescent dye for use with LIF detection/ 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQCA), has been recently developed for the CE-SDS method. It is a fluorogenic dye. Sample preparation is simpler than other traditional fluorescent labeling procedures, and the assay is easy to perform. 

Red. The spectra of XRITC and Texas Red are shifted to longer wavelengths compared to those of other rhodamines, which makes them particularly useful for dual labeling procedures in comhination with fluorescein (see Subheading 5.). Of the two, Texas Red, which is more hydrophilic and less likely to precipitate proteins upon conjugation (12), is more commonly employed. 

Fig. 1. Flowchart of the surface labeling procedure. Abbreviations as in text.

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

The cell labeling procedure described above appears to be of general applicability. We have tested it on several tumor cell lines obtaining invar-iantly a very efficient uptake with no apparent cytotoxicity. Likely, the entrapment of [GdHPD03A(H20)] into the endosomic vesicles prevents any impact of the paramagnetic agent on relevant cellular processes while maintaining the full accessibility to cytoplasmatic water molecules. 

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