Double labelling

Nohle U, Beau JM, Schauer R (1982) Uptake, metabolism and excretion of orally and intravenously administered, double-labeled A-glycoloylneuraminic acid and single-labeled 2-deoxy-2,3-dehydro-A-acetylneuraminic acid in mouse and rat, Eur J Biochem 126 543-548 Oxford J (2005) Oseltamivir in the management of influenza. Expert Opin Pharmacother 6 2493-2500... 

In order to obtain more insights into the biosynthetic mechanism, [Me- C-Me-d ] double-labeled methionine was fed to the organism. The C-NMR spectrum of resulting neosaxitoxin showed a clean triplet for C-13 beside the natural abundance singlet. The result indicated that only one deuterium was left on the methylene carbon. 

In another experiment, [l,2- C2-2-dJ double-labeled acetate was fed. First we observed a complete loss of deuterium atoms. In a short incubation, however, we obtained neosaxitoxin partially retaining a deuterium atom (40% equivalent of incorporated acetate molecule). The location of the deuterium atom was on C-5, which was originally the carboxyl carbon of acetate, suggesting that it migrated from the adjacent methyl-derived carbon C-6. 

Kure K, Lyman WD, Weidenheim KM, Dickson DW (1990a) Cellular localization of an HlV-1 antigen in subacute AIDS encephalitis using an improved double-labeling immunohistochemi-cal method. Am J Pathol 136(5) 1085-1092... 

Chattopadhayaya,. An NMR conformational smdy of the complexes of C/ H double-labeled 2 -deoxynucleosides and deoxycytidine kinase (dCK)./. Chem. Soc. Perkin. 

Recurrent is the lack of adequate techniques to assess carbon flows through the plants and microbes into soil organic matter (151). Most important is the development of techniques and protocols to separate rhizosphere from nonrhizosphere soil as well as possibly to facilitate analyses of soil carbon dynamics. The use of carbon isotopes, and, where possible, application of double labeling with C and C, seems inevitable in order to separate the contribution of different substrates to the formation of the soil organic matter pool and to get to an understanding of the ecological advantage of exudates and rhizodeposits. 

To distinguish adjacent 13C labels from natural abundance isotopes, proton-detected 13C-NMR spectra (HMBC) will show cross peaks associated with the double label that are split into doublets. In contrast, natural abundance 13C will show single cross peaks. 

FIGURE 7.11 13C-NMR spectrum of d(CGCAAATTTGCG)2 reductively alkylated with the 3a,3-13C double-labeled cleaving agent shown in the inset of Scheme 7.12. 

With a 13C label at the methide center, the presence of reactive methide intermediate can be verified and complex reaction products can be inventoried and eventually identified. The only limitations are the synthesis and cost involved in incorporation of the 13C label. As a rule we, only use 13C-labeled dimethylformamide and NaCN as starting materials because of their low cost and availability. Another limitation of enriched 13C-NMR monitoring is dilution of the enriched label to natural abundance levels. Currently, we are developing isotope-editing techniques that utilize unnatural 13C double labels to solve this problem. 

Figure 1.2 Regression analysis between numbers of C-Fos IR + GAD double-labeled neurons and the amount of sleep in the 2 h preceding sacrifice. The numbers of double-labeled cells were highly correlated with the percentage of preceding sleep in VLPO and both rostral and caudal MnPN. From Gong et al. (2004b).

Figure 1.6 (A) Changes in c-Fos IR in hypocretin/orexin(HCRT+)- containing neurons in response to BIC treatment as a function of distance from the microdialysis probe. The percentage of double-labeled cells was greatest closer to the probe (grids 1 and 2, compared with 3 and 4) and was greater with higher concentrations or exposure times. Contralateral cells were not affected. (B) Much lower percentages of melanin-concentrating hormone (MCH+) exhibited c-Fos IR activation in response to BIC. From Alam et al. (2005).

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. 

Luppi, P. H., Sakai, K., Fort, P., Salvert, D. Jouvet, M. (1988). The nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat nucleus reticularis magnocellularis a double-labeling study with cholera toxin as a retrograde tracer. J. Comp. Neurol. 277, 1-20. 

Rizzuto, R., Brini, M., De Giorgi, F., Rossi, R., Heim, R., Tsien, R. Y. and Pozzan, T. (1996). Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr. Biol. 6, 183-8. 

In a similar fashion, steroids are molecules that have been investigated by disruption of FRET. The sensor is a double labeled peptide with cyclodextrin bound to one side chain. The latter keeps the fluorophores closely together by accommodating the coumarin into its cavity thereby ensuring efficient FRET. Steroids compete for the cavity of cyclodextrin and displace the coumarin reducing FRET efficiency. This model, although useful for in vitro applications, seems to be poorly selective for its application in biological samples [95],... 

Okamura, Y. and Watanabe, Y. (2006). Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals. Methods Mol. Biol. 335, 43-56. 

Ino H. Application of antigen retrieval by heating for double-label fluorescent immunohistochemistry with identical species-derived primary antibodies. J. Histochem. Cytochem. 2004 52 1219-1230. 

Double-label ICC is another approach using a similar principle. It pairs a nuclear antibody with a cytoplasmic antibody on the same sample, such as keratin and estrogen receptor. At present, this method is mostly used in the research field. 

Scheme 12. Double-labelling experiments demonstrating concurrent alkylation and dealkylation in...

Aminomethylcoumarin derivatives possess intense fluorescent properties within the blue region of the visible spectrum. Their emission range is sufficiently removed from other common fluorophores that they are excellent choices for double-labeling techniques. In fact, coumarin fluorescent probes are very good donors for excited-state energy transfer to fluoresceins. 

Roth, J., and Binder, M. (1978) Colloidal gold, ferritin, and peroxidase as markers for electron microscopic double labeling lectin techniques./. Histochem. Cytochem. 26, 163. 

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