Affinity membrane chromatography for the analysis and purification of proteins

Affinity chromatography is unique among separation methods as it is the only technique that permits the purification of proteins based on biological functions rather than individual physical or chemical properties. The high specificity of affinity chromatography is due to the strong interaction between the ligand and the proteins of interest. Membrane separation allows the processing of a large amount of sample in a relatively short time owing to its structure, which provides a system with rapid reaction kinetics. The integration of membrane and affinity chromatography provides a number of advantages over traditional affinity chromatography with porous-bead packed columns, especially with regard to time and recovery of activity. This review gives detailed descriptions of materials used as membrane substrates, preparation of basic membranes, coupling of affinity ligands to membrane supports, and categories of affinity membrane cartridges. It also summarizes the applications of cellulose/glycidyl methacrylate composite membranes for proteins separation developed in our laboratory. (C) 2001 Elsevier Science B.V. All rights reserved.

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Introduction to Molecular Analysis of Ectomycorrhizal Communities

A number of methods are available for those researchers considering the addition of molecular analyses of ectomycorrhizal (EcM) fungi to their research projects and weighing the various approaches they might take. Analyzing natural EcM fungal communities has traditionally been a highly skilled, time-consuming process relying heavily on exacting morphological characterization of EcM root tips. Increasingly powerful molecular methods for analyzing EcM communities make this area of research available to a much wider range of researchers. Ecologists can gain from the body of work characterizing EcM while avoiding the requirement for exceptional expertise by carefully combining elements of traditional methods with the more recent molecular approaches. A cursory morphological analysis can yield a traditional quantification of EcM fungi based on tip numbers, a unit with functional and historical significance. Ectomycorrhizal root DNA extracts may then be analyzed with molecular methods widely used for characterizing microbiota. These range from methods applicable only to the simple mixes resulting from careful morphotyping, to community-oriented methods that identify many types in mixed samples as well as provide an estimate of their relative abundances. Extramatrical hyphae in bulk soil can also be more effectively studied, extending characterization of EcM fungal communities beyond the rhizoplane. The trend toward techniques permitting larger sample sets without prohibitive labor and time requirements will also permit us to more frequently address the issues of spatial and temporal variability and better characterize the roles of EcM fungi at multiple scales.

Failure of the plasma-sprayed coating of lanthanum hexaluminate

Lanthanum magnesium hexaluminate (LaMgAl11O19, LMA) is an attractive material for thermal barrier coatings (TBCs), and the failure of its coating was studied in this work by thermal cycling, X-ray diffraction, dilatometric measurement and thermal gravimetric-differential thermal analysis. The dilatometric measurement indicates that even though the bulk material of LMA has a higher sintering-resistance than the typical TBC material, i.e. yttria-stabilized zirconia (YSZ), the plasma sprayed coating of LMA has two serious contractions due to the re-crystallization of LMA and phase transitions of alumina.

Gold nanoparticle-based near-infrared fluorescent detection of biological thiols in human plasma

In this work, a new fluorescent method for sensitive detection of biological thiols in human plasma was developed using a near-infrared (NIR) fluorescent dye, FR 730. The sensing approach was based on the strong affinity of thiols to gold and highly efficient fluorescent quenching ability of gold nanoparticles (Au NPs). In the presence of thiols, the NIR fluorescence would enhance dramatically due to desorption of FR 730 from the surfaces of Au NPs, which allowed the analysis of thiol-containing amino acids in a very simple approach. The size of Au NPs was found to affect the fluorescent assay and the best response for cysteine detection was achieved when using Au NPs with the diameter of 24 nm, where a linear range of 2.5 x 10(-8) M to 4.0 x 10(-6) M and a detection limit of as low as 10 nM was obtained. This method also demonstrated a high selectivity to thiol-containing amino acids due to the strong affinity of thiols to gold.

Metabolic profiling of serum from gadolinium chloride-treated rats by H-1 NMR spectroscopy

Metabolic profiling of serum from gadolinium chloride (GdCl3, 10 and 50 mg/kg body weight, intraperitoneal [i.p.])-treated rats was investigated by the NMR spectroscopic-based metabonomic strategy. Serum samples were collected at 48, 96, and 168 h postdose (p.d.) after exposure to GdCl3. H-1 NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The studies showed that there was a dose-related biochemical effect of GdCl3 treatment on the levels of a range of low-molecular weight compounds in serum. The liver damage induced by GdCl3 was characterized by the elevation of lactate, pyruvate, and creatine as well as the decrease of branched-chain amino acids (valine and isoleucine), alanine, glucose, and trimethylamine-N-oxide concentration in serum samples. The biochemical effects of GdCl3 in rats could be consulted when evaluating the biochemical profile of gadolinium-containing compounds that are being developed for nuclear magnetic resonance imaging.

Short-capillary electrophoresis with electrochemiluminescence detection using porous etched joint for fast analysis of lidocaine and ofloxacin

Fast analysis of ofloxacin and lidocaine, as bactericide and analgesic or anesthetics, is of clinic importance for understanding the patient's medical process. This paper presented a high throughput, simple analysis method of lidocaine and ofloxacin by capillary electrophoresis coupled with electrochemiluminescence (ECL) using porous etched joint. To shorten the analysis time and to improve the analytical performance, a capillary with 10 cm in length was used as the separation channel. The cyclic voltammograms of Ru(bpy)(3)(2+) with different capillary length at same field strength showed that the porous etched joint eliminated the effect of electrophoretic current on the ECL detection. Following micro total analysis systems (muTAS), some advantages of which this approach has, the fabrication of channel in chip was not needed. Compared with capillary electrophoresis with 40-cm-long capillary, the high sample throughput and low zone broadening may be the main advantage of the present system. Under optimal condition, the detection limits of lidocaine and ofloxacin based on peak height were 3.0 x 10(-8) and 5.0 x 10(-7) mot L-1 and a 60 h(-1) of sampling frequency was obtained.

NMR and pattern recognition studies on the acute biochemical effects of Lu(NO3)(3)

Pattern recognition methods were applied to the analysis of 600 MHz H-1 NMR spectra of urine from rats dosed with compounds that induced organ-specific damage in the liver and kidney. Male Wistar rats were separated into groups (n=4) and each was treated with one of following compounds: HgCl2, CCl4, Lu(NO3)(3) and Changle (a kind of rare earth complex mixed with La, Ce, Pr and Nd). Urine samples from the rats dosed with HgCl2, CCl4 and Lu(NO3)(3) were collected over a 24 h time course and the samples from the rats administrated with Changle were gained after 3 months. These samples were measured by 600 MHz NMR spectroscopy. Each spectrum was data-processed to provide 223 intensity-related descriptors of spectra. Urine spectral data corresponding to the time intervals, 0-8 h (HgCl2 and CCl4), 4-8 (Lu(NO3)(3)) h and 90 d (Changle) were analyzed using principal component analysis (PCA). Successful classification of the toxicity and biochemical effects of Lu(NO3)(3) was achieved.

Investigation on acute biochemical effects of Ce(NO3)(3) on liver and kidney tissues by MAS H-1 NMR spectroscopic-based metabonontic approach

High resolution magic angle spinning (MAS)-H-1 nuclear magnetic resonance (NMR) spectroscopic-based metabonomic approach was applied to the investigation on the acute biochemical effects of Ce(No-3)(3). Male Wistar rats were administrated with various doses of Ce (NO3)(3)(2, 10, and 50 mg(.)kg(-1) body weight), and MAS H-1 NMR spectra of intact liver and kidney tissues were analyzed using principal component analysis to extract toxicity information. The biochemical effects of Ce (NO3)(3) were characterized by the increase of triglycerides and lactate and the decrease of glycogen in rat liver tissue, together with an elevation of the triglyceride level and a depletion of glycerophosphocholine and betaine in kidney tissues. The target lesions of Ce (NO3)(3) on liver and kidney were found by MAS NMR-based metabonomic method. This study demonstrates that the combination of MAS H-1 NMR and pattern recognition analysis can be an effective method for studies of biochemical effects of rare earths.

Determination of ultra-trace concentrations of elements in high purity tellurium by inductively coupled plasma mass spectrometry after Fe(OH)(3) coprecipitation

In this work, a method was established for the determination of impurities in high purity tellurium by inductively coupled plasma mass spectrometry (ICP-MS) after Fe(OH)(3) coprecipitation. After comparison of coprecipitation ability and separation efficiency between Fe(OH), and Al(OH)(3), Fe(OH)(3) was chosen as the precipitate. A separation factor of 160 for 200 mg tellurium was obtained under conditions of pH 9 and 2 mg of Fe3(+). The 13 elements, such as Bi, Sn, Pb, In, Tl, Cd, Cu, Co, Ni, Zn, Ti, Be and Zr, could be almost completely coprecipitated under these conditions. In addition, Te memory effect imposed on the ICP-MS instrument was assessed, as well as Te matrix effect that caused the low recovery of Ga, As, Sb and V in real sample was discussed. Finally, the method was evaluated through recovery test and was applied to practical sample analysis, with detection limits of most of the elements being below 0.15 mug g(-1) and R.S.D. below or at approximately 10%, which indicated that this method could fully satisfy the requirements for analysis of 99.999% similar to 99.9999% high purity Te.

Evaluation of bioeffects of a long-term administering ChangLe on rat by proton NMR method and biochemical examination

High resolution H-1 nuclear magnetic resonance ( NMR) spectroscopy has been employed to assess long-term toxicological effects of ChangLe (a kind of rare earth complex applied in agriculture). Male Wistar rats were administrated orally with ChangLe at doses of 0, 0.1, 0.2, 2.0, 10 and 20 mg/kg body weight daily, respectively, for 6 months. Urine was collected at-day 30, 60, go and serum samples were taken after 6 months. Many low-molecular weight metabolites were identified by H-1 NMR spectra of rat urine. A decrease in citrate and an increase in ketone bodies, creatinine, DMA, DMG, TMAO, and taurine in the urine of the rats. receiving high doses were found by H-1 NMR spectra. These may mean that high-dosage of ChangLe impairs the specific region of liver and kidney, such as renal tubule and mitochondria. The decrease in citrate and the increase in succinate and alpha-ketoglutarate were attributed to a combination of the inhibition of certain citric acid enzymes, renal tubular acidosis and the abnormal fatty acid catabolism. The information of the renal capillary necrosis could be derived from the increase in DMIA, DMG and TMAO. The increase in taurine was due to hepatic mitochondria dysfunction. The conclusions were supported by the results of biochemical measure. merits and enzymatic assay.

Major, minor and trace-elemental contents analysis in northeast soybeans by ICP-AES

Major, minor and trace elemental contents in northeast China soybeans were determined by using inductively, coupled plasma atomic emission spectrometry (ICP-AES). Three different sample digestion methods including two wet digestions, HNO3-HClO4 and HNO3-H2SO4 and a dry ash method were compared. Owing to the high oil content in soybeans, long time is needed and access acid should be added, with mixed acid digestion methods, which may result in higher sample blank. Therefore, the dry ask method would be more proper for the pre-treatment of soybean samples. Potassium and phosphorus are major elements in soybeans, so the effect of potassium and phosphorus on the other elements was investigated. Results showed that the potassium and phosphorus did not affect the determination. of other trace elements. There are not significant differences in trace elemental contents for the eleven northeast China soybeans.

Matrix effects in inductively coupled plasma mass spectrometry by use of organic solvents

Matrix effects arising from ethanol, propanol, glycerol, acetic acid, ethylenediamine and triethanolamine in inductively coupled plasma mass spectrometry have been studied. Addition of ethanol, propanol, glycerol, acetic acid, ethylenediamine and triethanolamine into solution has an enhancement effect on the signal intensity of analyte with ionization potential between 9 and 11 eV. The ethylenediamine and triethanolamine have higher enhancement effect on the signal intensity of Hg than that of ethanol, propanol, glycerol and acetic acid. Addition of ethylenediamine and triethanolamine into solution has a suppression effect on the signal intensity of Ph and Sr. The mechanism of the enhancement or suppression was investigated. The signal enhancement of Hg in the presence of ethylenediamine and triethanolamine is not caused by improved degree of ionization of Hg and nebulization efficiency. The suppression effects of Ph and Sr in the presence of ethylenediamine and triethanolamine are due to decrease of atomization efficiency of these elements. A method for the determination of Hg in the biological standard samples Ly ICP-MS was developed.

Determination of ultra-trace rare-earth elements in human plasma by inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS),a highly sensitive inorgnic analytic technique,fits to determine ultra-nace rare-earth elements in human plasma. Under the optimized conditions detection limits for 15 rare-earth elements are in the range of 0.7 (for Eu)-5.4 (for Gd) ng.L-1. Indium as an internal standard element is used to compensate for matrix suppression effect and sensitivity drift. Three kinds of preparation methods, diluted with 1% HNO3, digested with HNO3-H2O2 and with HNO3-HClO4, are checked and compared,and the former is the simplest way to be measured. The samples diluted with 1% HNO3, stored in 4 degrees C, are very steady for 16 days. With the method, 11 healthy plasma samples in Changchun area of China are analysed.