Radioactive methods, counting mixtures

To determine the efficiency of aminoacylation of [14C]Phe-tRNA, 5 fil aliquots of the aminoacylation mixture are withdrawn before and after the reaction the samples taken from the reaction mixture at the end of the incubation are spotted onto 3-MM paper discs (Schleicher Schuell) and processed by the cold TCA precipitation method, while the sample taken before the reaction is spotted on a paper disc pretreated empty by the same cold TCA procedure. Determination of the radioactivity present on these filters by liquid scintillation counting allows one to calculate the aminoacylation efficiency of the reaction (which, for phenylalanine, should be >2% of total tRNA). The specific activity of the [14C] Phe-tRNA can be determined after one-step purification of Phe-tRNA by BD cellulose chromatography (Gillam et al., 1968), followed by determination of the radioactivity and of the A260. 

Method. 1 mmole of compound is added to 1 ml of anhydrous pyridine in a S-ml flask. 2 mmoles of [I4C]acetic anhydride are then added for each expected reactive group (e.g., hydroxyl). The resulting mixture is heated at 12S °C for 4 h, cooled and poured into SO ml of cold water. This solution is transferred to a separating funnel and extracted with SO ml of diethyl ether. The diethyl ether is then washed three times with dilute hydrochloric acid, and once with each of dilute sodium hydroxide, water and saturated sodium chloride. The diethyl ether is dried with anhydrous sodium sulfate and evaporated to dryness for radioactive counting (using a proportional-flow counter or a scintillation counter). 

The reaction was stopped by adding 25 pi EDTA and 25 pi TCA (trichloroacetic acid). The reaction mixture was then spotted on ionic paper (DEAE paper). The paper was washed three times with TCA and then with ethyl alcohol. The filter paper was air dried and put into a scintillation vial with a scintillation cocktail. Radioactivity was measured by a liquid scintillation counter (Blue Star). As a counting control, a blank silver composition was run through the complete procedure without viral load, to check any potential interference in the scintillation counter method. 

Endogenous HBV polymerase assays measure the ability of the viral polymerase to incorporate dNTPs into viral replicative intermediates present within viral core particles. If the cores are derived from secreted virions, the assay measures predominantly DDDP activity, whereas if the particles are isolated from cytosol of infected cells, the assay measures predominantly RDDP activity. In these assays, one dNTP is usually radiolabeled, and polymerase activity is detected by scintillation counting of radioactivity incorporated into acid-precipitable material, presumed to be HBV DNA. In most published assay methods, a large excess of unlabeled dNTPs is present (see Note 1). In addition to dNTP substrates, reaction mixtures used for polymerase assays typically contain a buffer (usually Tris-HCl, pH 7.5-8.0), a monovalent salt (usually KC1), essential divalent metal ions (Mg2+ or Mn2+), a reducing agent (dithiothreitol [DTT] or 2-mercaptoethanol), and a nonionic detergent (usually Nonidet-P40 or Triton X-100). Concentrations of individual components of the reaction mixture in different published assay methods vary considerably. 

Competition methods are employed for ratios of less than 5-10% when two isotopic species react at different rate a mixture of the two will change composition (except at 100% reaction) and yield a product having a different isotopic composition from the reactant. This will give us a measure of the isotope effect. Radioactive isotopes give specific radiation (counts/sec/mole) proportional to the ratio of radioactive over stable isotope radioactivity may be measured with a precision better than 1%. 

When some commercial surfactants exhibited a precipitous drop in and efficiency between 0.5 and 5% water content, as did the nonionic surfactants Triton X-100 or Triton N-101, others behaved in a manner similar to our contrived nonionic-anionic surfactant mixtures. It is thus apparent that accurate calculation of the relative radioactivity of emulsified aqueous samples depends on rigorous uniformity of sample preparations. Optimal proportions of surfactants, surfactant content, and water content will depend on the nature and amount of the solute being counted. The usual methods of quench correction in LSC must be examined very carefully, inasmuch as they will usually not be adequate for determining absolute counting efficiency. 

Following an incubation at the time and temperature appropriate to each assay, reactions are terminated in order to separate L not bound to the R. This is done using one of two methods, centrifugation or filtration. In the former, the incubation mixtures are centrifuged at high speed (e.g., 50 000 10 min) to pellet the tissue. The incubation solution is decanted and the pellet is washed rapidly with 5-10 ml of ice-cold assay buffer. The pellet is dissolved in tissue solubilizers, scintillant is added, and the radioactivity contained in the pellet is counted using scintillation spectrophotometry. 

Until recently, this has been one of the most useful of the isotope dilution methods because it allows the quantitative conversion of a small amount of an unlabelled compound to an isotopically labelled derivative. The unlabelled compound is reacted quantitatively with a radioactive agent of known specific activity and the radioactive derivative is isolated, purified and counted. The activity recorded indicates the amount of radioactive reagent it contains and since the stoichiometry of the reaction is known the amount of compound present can be calculated. There are two basic requirements. First, the compound to be analysed must be quantitatively converted in high and reproducible yield to the radioactive derivative second, there must be a good method of isolating and purifying the labelled derivative. The first experiments to use an isotope derivative technique focused on the determination of amino acids in a mixture [263-265]. The reagent used was I-pipsyl chloride (/ -iodobenzenesulphonyl chloride). 

The results of the two experiments performed to date are given in Table 4. All samples constituting one complete experiment were counted in one 24-hr period, thus correcting for S radioactive decay. The different radioactive compositions of the two incubation mixtures are due only to matters of laboratory convenience, and do not reflect changes in chemical composition. So far as an illustration of the counting method, it can be seen that 88-89% of... 

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