Labeling Mixtures

If a mixture contains more than one chemical capable of causing a serious chronic effect, the potential effects of each should be combined in one statement of hazard. 

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Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

Labeling mixture For each reaction, make up 250 pL staining buffer, 0 1 pg antidigitoxin-FITC conjugated antibody (Boehnnger Mannheim). Use immediately. 

The protein-label mixture is gently mixed by rotating at room temperature for 15 min. 

The protein-label mixture is applied onto the column and... 

The stability of the preparations was evaluated after labelling the DOTATATE with and Lu with low and high activities. After SepPak purification, the labelling mixtures were stored under refrigeration in a reaction medium and in ethanol (96%). The stability of the radioiodinated DOTATATE and TATE was also evaluated after incubation of 100 j.L of each preparation (37 MBq), in triplicate, in 2.0 mL of human plasma at 37°C for 1,4 and 24 h. 

FIG. 16.7. HPLC purification of Lu-OCT (a) UV detection at 220 nm, (b) gamma detection of the labelling mixture and (c) control of the radiochemical purity of the radiopeptide after further purification by SepPak. 

Among all the aspects of MS/MS, isotopic assay should be mentioned. If the molecular ion is sufficiently abundant [194] then the conventional mass spectrum can be used to analyse the labeled molecules. However, if an isomeric labeled mixture is present it becomes difficult to attribute total deuterium for each of these species, as well as localization. For instance, the classical method cannot be used for the study of the mixture do, dj and d2 of phenyl-2 ethanol a and phenyl-1 ethanol b. The decomposition ions in the source are not sufficiently specific (randomization of deuterium). The m/z 92 and m/z 107 ions are characteristic decompositions of a and b compounds (Fig. 41). 

Fig. 4. Starch gel electrophoretic patterns and curve.s of radioactivity of labeled mixtures of Triton and a-LP. A a-LP-I i (2 mg), specific activity 0.5 pC/mg. B mixture of a-LP-PSt (2 mg) and Triton (40 mg) C Triton-I Si, specific activity 0.2 M-C/mg D mixture of a-LP (2 mg) and Triton-Ii i (40 mg). P = protein-stained pattern L = lipid-stained pattern (from Scanu and Oriente, 1961).

Incubate on a 50-)iil drop of run-on DNA labeling mixture, containing (per 100 fi ) 50 pd DNA synthesis buffer (twofold concentrated) plus 25 fi DNA labeling mixture and 25 /il water, for 3 to 30 min. The labeling time may be chosen so that a good fluorescent signal is obtained. Subsequently, wash the cells on 100 /i.1 TBS supplemented with 0.05% Triton X-100 for 3 min, followed by 3 min on 100 /tl TBS without detergent. 

Usually 100 - 500 ng of probe DNA are labeled. The DNA is denaturated in 15[il of destilled water by boiling for 10 min. After a short chilling on ice 2pl of a Hexanucleotide mixture, 2pl of a dNTP labeling mixture and finally Ipl Klenow enzyme (2 units) are added. The reaction is incubated for one to several hours at 37 C. In order to precipitate the DNA lOpl 3M NaAc, 70pl water and 300pl ethanol are added and kept 30 min at -20°C. After centrifugation for 15 min at 13.000 rpm the pellet is washed briefly with 70% ethanol, dried and redissolved in lOOpl TE - buffer (10 mM Tris-HCl ImM EDTA pH 7.8). 

Fig. 1. Stoichiometry of chlorine-containing silicides calculated from analysis data. (Results from Table 1, single silicide phases are labeled, mixture phases unlabeled).

Labeling Reaction. Labeling media were buffered at pH 7.2 to 7.4 and usually contained 6-20 vaM Mg, 80-150 mil/ NH4CI, and radioactively labeled aflSnity reagent at 10- to 100-fold excess over ribosomes added in the 70 S form or as isolated 30 S subimits (sometimes preactivated ). To enhance the specificity and stability of the reversible complex of oligonucleotide and ribosome, some reaction mixtures were supplemented with the cognate aminoacyl-tRNAs. > Labeling mixtures were usually incubated for 1-2 hr at 0°-37°. 

CarefuUy discard the labeling mixture according to regulations. 

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