Radioactive scintillation counting direct

Instead of scraping and manual collection of the adsorbent, the band can be sucked off the plate with a Vacuum-cleaner -type apparatus. Dekker [50] described an apparatus for the isolation of compounds from layers by elution and direct Millipore filtration, and Platt [51] designed a zone collector that used vacuum to transfer separated zones from layers direcdy to vials for hquid scintillation counting of radioactivity. 

Three aliquots from each (processed) sample were used for radioactivity determination. The aliquots from the urine samples were measured directly in the liquid scintillation counting procedure after addition of the scintillator Roth-rotiszint eco plus (Roth, Karlsruhe, Germany). The other matrix aliquots taken up on Combusto Cones (Canberra-Packard), weighed, dried at room temperature, were combusted in a Tricarb combuster (Canberra-Packard GmbH, Model 307) and the 14CC>2 formed was absorbed by Carbo-Sorb (Canberra-Packard). The subsequent radioactivity measurements were carried out by the liquid scintillation counting procedure in a ((-spectrometer (Canberra-Packard 2500 TR) after addition of the scintillator Permafluor E+ (Canberra-Packard). 

Determination of Radioactivity. All samples were counted in a Nuclear Chicago Isocap 300 liquid scintillation counter equipped with a Teletype computerized for direct calculation of disintegrations per minute. Fifty microliters of blood were directly counted for radioactivity after solubilization (1 mL of 1 N NaOH). After incubation at room temperature for 15 min the sample was decolorized by adding 200 fxL of hydrogen peroxide and incubating at 80°C for 30 min. The processed samples were mixed with 100 fxL of 80% acetic acid and 15 mL of Insta-gel (Packard), and were counted. Approximately 60-100 mg of tissue were solubilized following the same method as for blood. From the collected urine an aliquot (2 mL) was counted directly with 15 mL Insta-gel. The feces were dried overnight at room temperature, and a 60-100-mg aliquot was combusted (12) and counted for radioactivity. 

Quantitate the radioactivity, as a measure of RT activity in 1 pL of the culture supernatant, either by liquid scintillation counting (cut out the spots and place them directly into scintillation fluid), by phosphorimage analysis and quantitation of relative pixel units, or by autoradiography and laser densitometry. 

Each animal was sacrificed by captive bolt after the appropriate withdrawal interval (4, 6, 14 and 28 days after the 2nd dose) and processed as in an abattoir. The entire liver, kidneys and udder were excised and 1-2 kg samples of muscle from both the flank and the udder diaphragm and 1-2 kg samples of fat from the abdominal area were collected. Each organ and tissue was minced and processed three times through a commercial meat grinder to prepare respective homogenate samples. Sub-samples (200-300 mg) were prepared in triplicate for total residue analysis. Total radioactivity concentrations, expressed as pirlimycin free base equivalents, were determined by direct liquid scintillation counting (liquids) or combustion analysis (solids) following standard techniques. 

Bands can also be excised and radioactivity determined directly by scintillation counting. 

An alternative strategy for detection, where cellular systems or enzymatic fluxes are being investigated, is the use of radioactively labelled arachidonic acid or glutathione, which can function as precursor molecules and facilitate detection of the synthesised radio-labelled leukotrienes. The labelled molecules can be detected either directly by a radioactive flow monitor, or indirectly by fraction collection and scintillation counting. 

The wild-type (25)s insert is purified from the plasmid and end-labeled with Klenow fragment exactly as described for the Southwestern experiment, except that we start out with 50 /xg plasmid to get a final yield of approximately 2 /xg of insert. The 2 jxg of insert is labeled separately in two reactions, as described for Southwestern probe labeling. It is important that the probe has a high specific activity. After purification through Sephadex G50, radioactivity of the probe is measured by scintillation counting. Expect to get at least 2x10 cpm//xl, or 2 x 10 cpm in 100 /xl. Total counts are 4 x 10 in 200 /xl. Add directly to 100 ml hybridization buffer. 

Measurement Techniques. An aliquot portion of the radioactive solutions was directly added to the liquid scintillator, in the counting vials. As soon as prepared, these vials were shaken for 30 minutes (maximum speed of a rectilinear alternate shaker providing 280 shakes/minute), then counted. The measurements of each sample were repeated periodically. 

Liquid scintillation counting (LSC) is the usual method applied for the direct measurement of T radioactivity. Samples in which T is the major source of radioactivity are introduced into a scintillation cocktail. Prior conversion to water, followed by distillation to purify the sample from other radionuclides, is the preferred forensic method unless the analyst is certain that no other radionuclides are present. LSC on T-bearing aqueous specimens involves the use of an appropriate emulsifier to distribute the sample throughout the organic scintillator without degradation in performance. LSC calibration standards are available from NIST, and compensation for the efficiency change caused by the introduction of varying amounts of sample solution into the scintillator is discussed in Sansoni and Kracke (1971). 

The three main ways of detecting radioactive isotopes on TLC plates are (1) film registration or autoradiography, (2) zonal analysis (plate scraping and liquid scintillation counting), and (3) direct, in situ methods using radiation detectors. 

Finally, radioactively labeled retinoids can be detected through scintillation counting. This can be done either by the collection of fractions for determination of radioactivity in a standard liquid scintillation counter or by using a flowthrough detector, in which the sample eluting from the HPLC column is mixed with a scintillation fluid and then passed directly through a counting chamber. 

The principal methods for detecting and quantifying radioactivity on TLC plates are autoradiography, zonal analysis (plate scraping followed by liquid scintillation counting), and direct measurement using radiation detectors. The method employed for analysis depends on the available equipment, which generally depends on the amount of money available, and the type of experiment and information required. The various detection methods are discussed in outline below, and more detailed information can be obtained from the literature cited. 

Over the last 30 years or so the detection of radioactivity directly on TLC plates has taken dramatic leaps forward. Prior to the introduction of radiation detectors, the classical method used for the detection and quantitation of radioactivity on a plate involved exposure to x-ray film as the first step. This could take from a few hours up to one or two months, and this technique only located the radioactivity. The second step after location was quantitation, which was achieved by removing the zone of interest, either by scraping the silica gel off or by cutting away if the plates were aluminum- or plastic-backed, followed by liquid scintillation counting. Such a procedure is extremely labor intensive and is limited in terms of accuracy and resolution (see above). 

B. For radioactive antibiotic, after development, 5-mm wide strips of the silica gel were scraped off the plates directly into the counting vials and the radioactivity was determined by conventional scintillation counting techniques... 

In the estimation of unlabelled metabolites extracted from biological materials, enzymes have been used to convert non-radioactive substrates into labelled derivatives. A labelled co-factor is used in a manner similar to the use of a labelled reagent in isotope derivative analysis. A good example of such methods is the double isotope enzymatic assay for histamine [325]. Samples containing unknown amounts of histamine, tracer amounts of H-histamine and " C-S-adenosylmethionine are incubated with a partially purified preparation of histamine methyl transferase from guinea-pig brain. This enzyme is specific for the methylation of histamine [326]. The product of the reaction, 1,4-methylhistamine, is extracted into chloroform and the ratio of determined by liquid scintillation counting, is directly pro-... 

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