Degree of labeling

The nondestructive introduction of a fluorescent label would provide the molecule with a nonradioactive fluorophore, yet would preserve the option for direct radiolabelling of the fluorescent moiety with 125Iodine. This approach was pioneered by Nagasawa et al. (5) who reacted native or /V-desulfated heparins with a fluorescein isothiocyanate (FITC). The resulting degree of labelling was low... 

Figure 2. The pH dependency of heparin labelling. The degree of labelling intensity of the final product was compared with the concomitantly measured solubility of the F-D labelling reagent.

For amino acid analysis the labeled protein needs to be hydrolyzed and derivatized. Most commonly the hydrolysis is performed in 6 M HC1, and the amino acids are converted into their isopropyl ester and pentafluoropropanamide derivatives (Fig. 1.1) before GC/ MS analysis. The molecular ion is not always visible after standard electron impact (El) ionization, and the fragment after loss of the carboisopropoxy group is the highest observable peak. This leaves m/e=175 plus the mass of the amino acid side chain, from which the degree of labeling can be directly deduced. 

Degree of labeling of gluconic acid residues at present is unknown... 

The specific activities of the different radiomethylated milk proteins varied between 3 and 4.4 pC /mg. This degree of labeling, resulting from the use of 14C-HCHO of high specific activity, allows the proteins to be used as true tracers. For example, the addition of 1 mg of labeled protein with a specific activity of 4 /xCi/mg to a liter of milk should provide excellent counting statistics on less than 0.5 mL of the milk. Also in the experiments on the plasminolysis of /3-casein, the radioactive /3-casein was diluted 100-300-fold with cold /3-casein before experimenta-... 

Protein A is a specific protein isolated from the cell wall of Staphylococcus aureus whose characteristic property is the ability to react and to form precipitates with a variety of IgG molecules from several species. This interaction is reminia nt of the formation of antigen-antibody complexes, and has been used to study different aspects of immune response as well as cell surface structure and function For easier detection, Protein A is covalently coupled to fluorescein isothiocyanate (FITC). The commercial preparations of FITC-Protein A contain an avarage of 6 FITC substituent groups per molecule of protein. Such a degree of labeling does not affect the biological properties of the native protein. 

The degree to which a radioactive compound is labeled is given in terms of its specific activity (CPM//i,mole, mCi/mmole, and so on). The degree of labeling of a compound ivith a stable isotope is expressed in terms of atom percent excess. This term represents the proportion of stable isotope over and above that normally present in nature. For example, if P is the normal abundance (%) of a given isotope in nature and P, is the abundance (%) in a labeled (enriched) compound, then P, — P is the atom percent excess (APE). 

Fluorescent dye-polypeptide conjugates were prepared by labeling with either fluorescein isothiocyanate adsorbed onto Celite (51) or with 1-dimethylaminonaphthalene sulfonyl chloride (DNS) dissolved in ethanol (65). The degree of labeling was determined from the amount of dye on the polypeptide estimated by fluorescent intensity or ultraviolet absorption measurements and from the concentration of the polypeptide determined by Kjeldahl nitrogen. There were generally two to four dye residues per polypeptide molecule. 

The degree of labeling of a compound is expressed as the atom percent excess. Suppose that an element normally contains x atoms of a particular isotope per 100 atoms of the element the normal abundance would be x atoms percent, and there would be 100 — x atoms of other isotopes per 100 atoms. Suppose that the abundance is increased to y atoms percent by the labeling process the difference, y x, is called the atom percent excess of the particular isotope. 

Determine the degree of labeling by measuring absorbance of the dye, calculating the dye concentration, and calculating the molar dye/protein ratio. Manufacturers usually specify the molar absorption coefficient of the dye. Concentration C of the dye can then be calculated as ... 

Phosphorylase has a relatively low rate of turnover, and as such, large doses of radiolabeled pyridoxine were needed to obtain an adequate degree of labeling. In addition, the need for serial sampling of the decay curve, using individual animals, introduced substantial biological variation. Both aspects of this experimental system precluded application of the method to humans, and we considered the possibility of a different approach, based on stable-isotope-labeled pyridoxine, to monitor phosphorylase degradation. 

The terms and are proportional at low and modest extents of mixing [52], and the proportionality constant depends upon the degree of labelling of the sample and the ratio of donor- to acceptor-labelled particles [52,53]. In other words, if the acceptor concentration in the sanq/le is hi fmit) - 1 faster than /s(t) - 1 and if it is low, /m(t) - 1 more slowly than /s(r). 

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