Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.     

“同时蒸馏-萃取”分析茉莉花香成分

用一种经过改进的“同时蒸馏-萃取”仪提取了茉莉花的香成分,以 GC/MS 和 Kovats保留指数法鉴定了提取物中的28个化学成分。主要成分为:芳樟醇、乙酸苯甲酯、顺-石竹萜烯、榧烯醇、苯甲酸顺-3-己烯酯、邻氨基苯甲酸甲酯、二十三烯-11及吲哚。讨论了鲜花香成分和植物精油的采集与分析方法。     

Morphological evidence for the existence of a more complex cytoskeleton in Amoeba proteus

Summary Various stabilization and extraction procedures were tested to demonstrate the ultrastructural organization of the cytoskeleton in normal, locomoting Amoeba proteus. Most reliable results were obtained after careful fixation in glutaraldehyde/lysine followed by prolonged extraction in a polyethylene glycol/Triton X-100 solution. Before dehydration in a graded series of ethanol and critical-point drying, the amoebae were split by the sandwich-technique, i.e., by mechanical cleavage of cells mounted between two poly-L-lysine-coated glass slides. Platinum-carbon replicas as well as thin sections prepared from such cell fragments revealed a cytoskeleton composed of at least four different types of filaments: (1) 5–7-nm filaments organized as a more or less ordered cortical network at the internal face of the plasma membrane and probably representing F-actin; (2) 10–12-nm filaments running separately or slightly aggregated through the cytoplasm and probably representing intermediate filaments; (3) 24–26-nm filaments forming a loose network and probably representing microtubules; and (4) 2–4-nm filaments as connecting elements between the other cytoskeleton constituents. Whereas microfilaments are responsible for protoplasmic streaming and other motile phenomena, the function of intermediate filaments and cytoplasmic microtubules in amoebae is still obscure.     

Ion-pair extraction of bile acids with Lipidex gel

A new method for the extraction of bile acids from aqueous solutions, urine, plasma, and bile is described. A buffered solution of decyltrimethylammonium bromide is added to the sample to give a 0.03 m concentration of the counter-ion. The mixture is passed through a bed of Lipidex 1000, which is then washed with the buffered solution of counter-ion followed by water. The decyltrimethylammonium salts of bile acids are sorbed by the Lipidex and are eluted with methanol. Recoveries of unconjugated, taurine- and glycine-conjugated, sulfated, and glucuronidated bile acids are close to 100%. Unconjugated bile acids can also be quantitatively extracted from aqueous solutions and urine after acidification with acetic acid.     

Radioimmunoassay for quantitative determination of morphine in capsules of Papaver somniferum

A radioimmunoassay (RIA) procedure for the determination of pmol quantitites of morphine in capsule samples of Papaver somniferum was developed. An antiserum developed against a conjugate of morphine-3-hemisuccinate-BSA was relatively specific for morphine and possessed moderated cross-reactivity with codeine and mild cross-reactivity with thebaine, but none with narceine, papaverine, or noscapine. The standard curve was linear over a range of 0.01–0.20 ng. This assay allows for the rapid, sensitive and precise determination of morphine in unpurified aqueous extracts of capsule samples. The amounts of morphine in the aqueous extracts determined by radioimmunoassay were validated by high performance liquid chromatography (HPLC). The two methods show a high correlation coefficient (r = 0.98) with no significant difference in determinations of morphine content by RIA and HPLC.     

Effects on microbial activity by extraction of indigenous cells from soil slurries

Abstract: Possible effects on the physiological activity and culturability of soil microorganisms by different soil dispersion procedures, and effects on activity caused by extracting bacteria from soil, were investigated. There was no apparent difference in cfu's with dispersion of a silty loam soil and a loamy sand soil with pyrophosphate as compared to dispersion in NaCl. Substrate-induced respiration was reduced in the silty loam soil, and methanol oxidation was reduced in the loamy sand soil with dispersion in pyrophosphate, and the soil pH was irreversibly increased by the treatment. Extracted bacterial fractions had lower numbers of culturable cells as percentage of the total number of bacteria in each fraction, lower respiration rates and no methanol oxidation activity as compared to the soil slurry both before and after extraction. The physiological activity was apparently not affected by the number of cells extracted. This indicates that the increased extraction rate of indigenous soil bacteria obtained by effective disruption of aggregates and detachment of cells from surfaces, only results in increased extraction of cells that have been physiologically changed as a result of the extraction process.     

High-throughput RNA extraction from plant samples based on homogenisation by reciprocal shaking in the presence of a mixture of sand and glass beads

We describe here a reliable high-throughput method for extraction of RNA from fresh or frozen plant tissue that obviates laborious and time-consuming homogenisation by mortar and pestle. The method is based on homogenisation by high-speed reciprocal shaking in presence of a mixture of inexpensive abrasive materials; i.e., quartz sand and glass beads. After homogenisation, the method follows a standard procedure for RNA extraction by phenol/LiCl. Yield and quality of RNA obtained by homogenisation with the sand/glass bead mix are identical to those obtained by mortar and pestle.     

Identification of single tiny seeds ofOrobanche using RAPD analysis

The tiny seeds of parasitic weeds of the genusOrobanche can be identified by using RAPD markers. A simple procedure for DNA extraction from single seeds, 10 μg each, followed by RAPD-PCR and using specific DNA markers, leads to species identification. Seeds of five different species could be identified using this method.     

Rapid authentication of animal cell lines using pyrolysis mass spectrometry and auto-associative artificial neural networks

Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures.     

Improving protein extraction yield in reversed micellar systems through surface charge engineering

The mechanism of extraction of rat cytochrome b(5) from water into a sodium dioctylsulfosuccinate (AOT) micellar organic phase was studied using protein engineering of surface charged residues. The extraction behavior of native cytochrome b(5) and modified proteins with substitutions of the type glutamic acid --> lysine at positions 44 (E44K), 56 (E56K), and 92 (E92K), was studied as a function of pH. The results indicate that an important mechanism of extraction is an electrostatic interaction of this protein with the negatively charged surfactant. We demonstrate that it is possible to improve extraction by engineering the protein surface charge, increasing the driving force responsible for the protein transfer to the micellar phase. (c) 1994 John Wiley & Sons, Inc.