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1.
Summary A procedure for counting p32 in plant tissues is presented. The method, based on the use of Cerenkov radiation, involves practically no sample preparation.
Plant tissue are placed into vials containing water or hexane and counted with a liquid scintillation counter.
Counts obtained, using this procedure were found to be linearily related to that obtained with a G.M. tube. The counting efficiency
was, however, higher with the proposed method.
The use of hexane is advantageous if leakage of p32 from the tissue is possible, or when higher counting efficiency is desireable. The use of different liquids may also enable
a discriminative count of different beta emitters. As suggested recently8 use of wavelength shifter may further increase efficiency of counting Cerenkov radiation. 相似文献
2.
K.D. Neame 《Analytical biochemistry》1978,91(1):323-339
The reliability of the channels ratio method for determining counting efficiency in liquid scintillation counting was investigated. It was found that the efficiency of counting gels, cloudy samples, two-phase samples, samples in which the radioactive material was precipitating, and samples on solid supports could not be reliably determined from a normal quench correction curve. A curve constructed from external standard channels ratios was unreliable when mixing different vial sizes and sample volumes, but one constructed from sample channels ratios was not. It was also found that variation in instrument performance can result in large errors unless samples and standards are counted together. Statistical error changed relatively little within the range of ratios 0.3 to 0.8. 相似文献
3.
Thomas F. Kellogg 《Analytical biochemistry》1983,134(1):137-143
The effect of sample vial type and sample composition on the ?erenkov count rate detected from 32P and 36Cl was studied using a liquid scintillation counter. When counting was done in the noncoincident mode, glass vials allowed higher counting efficiency than plastic vials. In the coincident mode light scattering caused by polyethylene and polyproplyene vials allowed higher counting efficiency than glass vials. Highest coincident counting efficiency was from plastic minivials in a glass carrier vial. Increased solute concentration in samples caused increased counting efficiency due to changes in the refractive index of the solution. This can cause significant counting efficiency changes with no sample channel ratio change in density gradient fractions. The use of wavelength shifters is shown to be inappropriate when the sample pH varies, as this can change the fluorescent properties of the shifters and thereby the observed count rate. 相似文献
4.
An improved scintillation cocktail of high-solubilizing power 总被引:55,自引:0,他引:55
A scintillation cocktail containing 25% Triton X-114 in xylene is considered for a broad range of scintillation counting applications. The cocktail gives good counting efficiencies for 3H (47%) and 14C (93%). It will accept up to 30% (v/v) aqueous sample. The scintillation fluid is also used effectively with samples which are difficult to solubilize, such as the degradation products from the solubilization of polyacrylamide gels. The cocktail can be formulated for less than $2.00 per gallon. 相似文献
5.
Jorma Viikari 《Analytical biochemistry》1975,63(2):566-571
A rapid and simple method for counting radioactivity in tissue samples containing [3H]- or [14C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems. 相似文献
6.
If emitters of energetic β-particles (such as 32P) are counted on solid supports in liquid scintillation systems, considerable distortion of the apparent energy spectrum of the β-particles can result if the material is placed on the bottoms or against the walls of vials. This results if the path length in the scintillation fluid available to a substantial proportion of the β-particles is short compared with their range. The phenomenon leads to reduced and variable efficiency in normal counting channels and to difficulties in double-labeling. 相似文献
7.
Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a 14C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from 14C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system). 相似文献
8.
A method for analysing 32P in small aqueous samples by measuring the ?erenkov radiation is described. Centering problems with small sample volumes are eliminated by placing the sample in a polystyrene tube in the scintillation vial. The detection efficiency is high, 54.5 ± 0.6% (2 SD) at a sample volume of 25 μl. The reproducibility is good and independent of the sample volume. The detection efficiency of 32P in polyacrylamide gel is shown to be as good as in water. 相似文献
9.
Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a 14C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from 14C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system). 相似文献
10.
The effect of silica gel on the recovery of radioactivity from 14C- and 3H-labeled lipids by liquid scintillation is analyzed. In the most unfavorable ease, when counting with a toluenefluor solution directly added to the vials containing the adserbed lipid, drastic reductions in the counting efficiency were found, which depended on the amount of silica gel, sample activity, and chemical nature of the lipid. For certain lipids like phosphatidylcholine, these effects were not completely overcome even by introducing water and detergents in the counting system. This paper intends to draw attention to the fact that these factors should be especially taken into account when comparing different lipids from thin-layer chromatograms. 相似文献
11.
D C Wigfield 《Analytical biochemistry》1974,59(1):11-15
A method for obtaining the true activity and counting efficiency of a 14C sample partially or completely adsorbed on the walls of a counting vial by liquid scintillation counting is presented. 相似文献
12.
A method for the liquid scintillation counting of precipitated protein from red cells in 0.1–1.0 ml of blood is described. Precipitate is incubated for 0.5 hr at 100°C with equal volumes of acetic acid, ethyl acetate, and hydrogen peroxide; an equal volume of hydrochlorie acid is then added, followed by a toluene/Triton X-100 scintillation mixture containing primary and secondary scintillators. Maximum counting efficiencies with precipitate from 0.2 ml of blood were 90% for 14C and 35% for 3H. Recovery of labeled amino acid was not less than 90%. Chemiluminescence decayed to not more than 15 cpm above background in 45 min. 相似文献
13.
An external-sample liquid scintillation (LS) counting for the gamma emitter 75Se has been developed. An expressly designed well-type LS vial and a 2,5-diphenyoxazole-1,4-bis(5-phenyl-2-oxazoyl)-benzene-xylene
solution containing 35% tertrabutylzinn allow 75Se to be counted in a standard LS counter with counting efficiency up to 43.2%, much higher than that of conventional LS counting
method. This external sample LS has a good count rate linearity and exhibits low background count rates.
After in vivo labeling with [75Se]selenite, 75Se distributions and the Se-containing proteins present in tissues of male rat were investigated by means of sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, external-sample LS and γ-detector. Eight Se-containing proteins or protein subunits
were detected to be Se-containing proteins or protein subunits in arterial wall, and their apparent molecular masses (Mr)
were 76.4, 67.0, 57.4, 30.3, 25.4, 22.7, 21.7, and 15.1 kDa, respectively. In addition, eight 75Se-labeled proteins (Mr: 66.8, 57.0, 43.1, 30.0, 24.8, 19.8, 18.0, and 14.8 kDa) were found in brain homogenates, and nine
75Se-labeled proteins (Mr: 117.0, 78.0, 66.6, 57.2, 43.0, 38.1, 25.0, 20.1, and 18.0 kDa) were detected in testis homogenates.
Some of them should be new biologically important selenoproteins that have not been identified so far. 相似文献
14.
Thomas F. Kellogg 《Analytical biochemistry》1982,120(2)
The optimal way to count aqueous samples by liquid scintillation counting is in a homogeneous solution. Technical limitations have previously made this difficult. Triton X-100 is a water-miscible liquid scintillant which counts 14C with 80% efficiency and 3H with 17% efficiency. It has a high flash point (over 300°F), is nonvolatile, and does not cause swelling or leaching when used in polyethylene vials. Liquid-scintillation counting cocktail using Triton X-100 as the sole scintillant (i.e., no toluene or xylene) does not have to be disposed of as a hazardous waste. The large aqueous sample capacity of a miscible cocktail, its safety, and ease of disposal make its use highly attractive for many applications. 相似文献
15.
Incubation of 14C-labeled substrates continues to be a widely used procedure in soil organic matter (OM) research due to its sensitiveness. When the labeling is found in liquid fractions (soil extracts, hydrolysates), 14C can be easily quantified by using an aliquot for scintillation counting. For this reason, converting a solid carbon sample into liquid form is a typical step for accurate 14C analysis. We have developed an alternative method to carry out this step, which uses standard glass hardware and does not require complex laboratory facilities. Carbon (both in organic or inorganic forms) is converted into CO2 within a reaction vessel connected to a Twisselmann’s extractor with an alkali trap inside. This forms an individual closed chamber (ICC) for each sample, thus eliminating the risk of cross-contaminations. The alkali solution adsorbs the evolved CO2 within the closed system, and the excess of pressure is easily overcome by the use of a balloon. We tested the procedure on a set of substrates and two contrasting soils, checking also the effect of different sample loads (from 20 to 160 mg C) on the CO2 recovery of the process. The percentage of carbon recovered into the alkali (i.e. the efficiency of the process) ranged from 92% for the inorganic C to 93–95% for the organic C method, the latter being sensitive to the amount of sample used for analysis. The ICC method can be successfully applied to analyze 14C-labeling in both carbonates and OM from solid samples, thus representing an alternative method to some established protocols, and it is suitable for substrates with low or very low 14C contents, in which high volumes of sample must be analyzed in order to guarantee representative results. 相似文献
16.
Gramicidin A, a linear peptide antibiotic, makes membranes permeable to alkali cations and hydrogen ions by forming transmembrane channels. We report here conductance and fluorescence energy transfer studies of channels containing two kinds of gramicidin. These studies of hybrid channels were designed to determine the number of molecules in a channel. The gramicidins studied were gramicidin A, dansyl gramicidin C, the p-phenylazobenzene sulfonyl derivative of gramicidin C (PABS4 gramicidin C), and the 4-(diethylamino)-phenylazobenzene-4-sulfonyl chloride derivative of gramicidin C (DPBS gramicidin C). The dansyl, PABS and DPBS groups were linked to the hydroxyl group of tyrosine 11 in gramicidin C. The single-channel conductance of PABS gramicidin C in planar bilayer membranes is 0.68 that of gramicidin A. Membranes containing both PABS gramicidin C and gramicidin A exhibit three kinds of channels: a pure gramicidin A, a pure PABS gramicidin C channel, and a hybrid channel with an intermediate conductance (0.82 that of gramicidin A). The dependence of the frequencies of these three kinds of channels on the mole fractions of gramicidin A and PABS gramicidin C in the membrane-forming solution fits a dimer model. Fluorescence energy transfer was used as a complementary means of ascertaining the frequency of hybrid channels. Dansyl gramicidin C was the fluorescent energy donor and DPBS gramicidin C was the energy acceptor. The efficiency of energy transfer between these chromophores in hybrid channels in liposomes was 75%. The relative quantum yield of the dansyl fluorescence was measured as a function of the mole fraction of DPBS gramicidin C. These fluorescence studies, like the single-channel conductance measurements, showed that there are two molecules of gramicidin in a channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. 相似文献
17.
László Bene Tamás Ungvári Roland Fedor László Sasi Szabó László Damjanovich 《Biophysical journal》2013
Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determining proximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolute fluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenching and sensitized emission. A crucial point is the determination of the scaling factor α responsible for balancing the different sensitivities of the donor and acceptor signal channels. The determination of α is not simple, requiring preparation of special samples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation (least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of α and the absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the second moments (variances and covariances) of the detected intensity distributions. A quadratic equation for α is formulated with the intensity fluctuations, which is proved sufficiently robust to give accurate α-values on a cell-by-cell basis in a wide system of conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly new approach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines, FT and LS174T. The figures show that whereas the common way of α determination fails at large dye-per-protein labeling ratios of mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer, the new approach can also be straightforwardly used with fluorescence microscopes. 相似文献
18.
The counting characteristics of six liquid scintillation counters are compared. Each instrument was tested to determine the figure of merit (), the change in percentage counting efficiency and external standard ratio over sample volumes from 1 to 20 ml, the reproducibility of chemical quench curves over sample volumes from 5 to 20 ml, and the effects of increasing sample activity on the actual and calculated counting efficiency. Tests were performed using both 3H and 14C. The results show that differences in counting efficiency are more important than maximum sensitivity (). All instruments demonstrated the same response to changes in sample volume. The external standard ratio varied with sample volume in all instruments except one. Quench curves were essentially volume independent, with two exceptions. This study demonstrates that periodic performance tests must be conducted to assure that these instruments are operating in an efficient and reproducible manner. 相似文献
19.
To assess the role of selection pressure in plant adaptation to saline environment, a hydroponic experiment was conducted on six Panicum antidotale Retz. populations collected from a wide range of habitats with varying selection pressure in the form of soil salinity. The soil electrical conductivity of six different habitats ranged from 3.39 to 19.23 dS m−1 and pH from 5.86 to 7.65. Plants of all populations collected from varying habitats were established in pots containing normal soil and allowed to grow for 6 months. Newly grown tillers from each plant were separated and 10 of them each formed a composite sample for a particular population. They were then transplanted in plastic containers each containing 10 l of half strength Hoagland's nutrient solution alone or with 150 mol m−3 NaCl. After 42 days growth in salt treatment, the populations collected form highly saline habitats proved to be more salt-tolerant compared with those from mild or non-saline habitats in terms of growth performance. The populations adapted to high salinity showed less decrease in leaf K+/Na+ and Ca2+/Na+ ratios under salinity stress. Moreover, under stress the salt-tolerant populations showed less reduction in photosynthetic capacity than the salt-sensitive populations. In addition, hyper-accumulation of organic solutes such as glycinebetaine and proline and thereby higher osmotic adjustment seemed to be associated with the higher degree of adaptability of the salt-tolerant populations to salt stress. From the data presented, it is plausible to conclude that selection pressure (soil salinity) must have been one of the important determinants bringing about the evolution of salt-tolerance trait in Blue Panic grass. 相似文献
20.
Franz-Josef Schmitt Bastian Thaa Cornelia Junghans Marco Vitali Michael Veit Thomas Friedrich 《BBA》2014
The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H+ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy. 相似文献