Double labelling technique

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. 

Remote double labelling techniques have been used successfully in the determination of enzyme KIEs (Kiick, 1991). A variant of this technique was applied to a nonenzymatic reaction by Matsson and co-workers (Axelsson et al., 1990). They determined the primary carbon and secondary deuterium KIEs for the SN2 reaction between methyl iodide and hydroxide ion in 50% dioxane-water at 25°C. The a-carbon KIE was determined by the UC method (Axelsson et al. 1987,1991). In this method, a mixture of substrate molecules labelled with UC (tm = 20.4 min) and 14C is used. The reactants and products... 

The double labeling technique, combined with the technique of collection of xylem tissue at different stages of differentiation, provides additional information on the distribution of specific lignin substructures such as condensed units in different morphological regions. 

The double-labeling technique is also useful for in vitro studies on the mechanism of dehydrogenative polymerization of monolignols. 

On the other hand, j8 particles from 14C will travel, on average, 10/m through emulsion, and so a thicker layer of emulsion while reducing resolution has the effect of increasing the number of grains produced. This has been made use of in double label techniques where DNA has been labelled with tritiated and 14C-labelled... 

Kainosho M, Tsuji T, Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution, Biochemistry, 21 6273-6279, 1982. 

Small isotope-effects can be detected by double-labeling techniques, in which the carbon skeleton is labeled with 14C, and the ratio of 14C to tritium is measured both in the substrate and the product. Care must be taken in the observation and interpretation of isotope effects determined from the hydrogen-isotope content of the product. Just as in non-enzymic reactions (see p. 154), discrimination against the substrate containing deuterium or tritium leads to an increase in the isotopic content of the substrate, and this decreases the apparent isotope-effect towards the end of the reaction. 

Room P, Postema E, Korf J (1981) Divergent axon collaterals of rat locus coeruleus neiu ons Demonstratiorr by a fluorescerrt double labeling technique. Brain Res 221 219—230. 

Apps R, Trott JR, Dietrichs E (1991) A study of branching in the projection from the inferior olive to the X and lateral c, zones of the cat cerebellum using a combined electro-physiological and retrograde fluorescent double-labelling technique. Exp. Brain Res., 87, 141-152. 

Competitive KIEs can reduce the uncertainty in prefactor isotope effects, and have been used to demonstrate tunneling in several enzymes [24, 36, 65]. As discussed above, the competitive, double-label, technique for measuring KIEs is inherently more precise than noncompetitive techniques, and can reduce the experimental uncertainty in the KIE on the Arrhenius prefactor and energy of activation. The use of tritium also provides multiple ratios Ah/At and Ad/At, which are helpful in resolving kinetic complexity [61]. It has been noted that a change in the rate-limiting step over the temperature range can lead to anomalous Arrhenius... 

DAB is probably the most versatile chromogen in immunohis-tochemistry. Its brown color can be modified when used in the presence of nickel-chloride (purple-black), cobalt-chloride (dark-blue) or copper sulfate (dark brown) (25). Also, DAB and its metallic complexes can be further contrasted to form a black precipitate with silver intensification (26). Immunoreactions resulting in electron-dense precipitates, such as immunogold, IGSS, and IPO/DAB-complexes, have also been used widely for double/ multiple antigen detection at the ultrastructural level (1, 27). For light microscopy, one of our preferred double labeling techniques... 

Also, the measured fluorescence signal may not be proportional to the surface concentration of protein. This has been observed during adsorption experiments using fluorescently-labeled 7-globulins (17) and sperm whale myoglobin (Mb) (19). Therefore, the TIRF apparatus must be properly calibrated to determine whether fluorescence intensity is indeed proportional to surface concentration. Some early calibration techniques must be viewed with caution because they were not performed under the same conditions as the protein adsorption experiments (20,21). More recently, a reliable calibration procedure that uses a double labeling technique has been developed (17). 

The possibility that dihydroxyacetone phosphate (DHAP), as well as glycerol phosphate, may serve as the initial acyl acceptor in the pathway has not been extensively studied in plants. M. I. Gurr and D. N. Brindley (unpublished results) used the double-label technique of Manning and Brindley... 

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