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Separation Steps (separation + step)
Selected AbstractsTotal analysis of endocrine disruptors in a microchip with gold nanoparticlesELECTROPHORESIS, Issue 18 2010Hui-Bog Noh Abstract The development of a simple, sensitive, and direct method for the total analysis of certain endocrine disruptors was performed by integrating preconcentration steps to a separation step on a microchip through the modification of the field-amplified sample stacking and field-amplified sample injection steps. To improve the preconcentration and separation performances, the preconcentration and separation buffers were modified with citrate-stabilized gold nanoparticles (AuNPs). For the detection of the separated samples, cellulose-dsDNA/AuNPs-modified carbon paste electrodes were used at the channel end. The experimental parameters affecting the analytical performances, such as the buffer concentration, water plug length, SDS concentration in the separation buffer, AuNPs concentration, preconcentration time, detection potential and electrode to channel distance, were examined. The detection limits of the test compounds were between 7.1 and 11.1,fM and that for 4-pentylphenol was 7.1 (±1.1) fM. Dynamic ranges were in the range from 0.15 to 600.0,pM. The experiments with real samples were performed to evaluate the reliability of the proposed method. [source] Myristyl and palmityl acylation of pI 5.1 carboxylesterase from porcine intestine and liverFEBS JOURNAL, Issue 4 2002Tissue, subcellular distribution Immunoblotting analyses revealed the presence of carboxylesterase in the porcine small intestine, liver, submaxillary and parotid glands, kidney cortex, lungs and cerebral cortex. In the intestinal mucosa, the pI 5.1 enzyme was detected in several subcellular fractions including the microvillar fraction. Both fatty monoacylated and diacylated monomeric (F1), trimeric (F3) and tetrameric (F4) forms of the intestinal protein were purified here for the first time by performing hydrophobic chromatography and gel filtration. The molecular mass of these three enzymatic forms was,estimated to be 60, 180 and 240 kDa, respectively, based on size-exclusion chromatography and SDS/PAGE analysis. The existence of a covalent attachment linking palmitate and myristate to porcine intestinal carboxylesterase (PICE), which was suggested by the results of gas-liquid chromatography (GLC) experiments in which the fatty acids resulting from alkali treatment of the protein forms were isolated, was confirmed here by the fact that [3H]palmitic and [3H]myristic acids were incorporated into porcine enterocytes and hepatocytes in cell primary cultures. Besides these two main fatty acids, the presence of oleic, stearic, and arachidonic acids was also detected by GLC and further confirmed by performing radioactivity counts on the 3H-labelled PICE forms after an immunoprecipitation procedure using specific polyclonal antibodies, followed by a SDS/PAGE separation step. Unlike the F1 and F4 forms, which were both myristoylated and palmitoylated, the F3 form was only palmitoylated. The monomeric, trimeric and tetrameric forms of PICE were all able to hydrolyse short chain fatty acids containing glycerides, as well as phorbol esters. The broad specificity of fatty acylated carboxylesterase is discussed in terms of its possible involvement in the metabolism of ester-containing xenobiotics and signal transduction. [source] MALDI-TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varietiesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2009alplachta Abstract A procedure for identification of malting barley varieties using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of ethanol-soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI-TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry-based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd. [source] Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix-assisted laser desorption/ionization-based proteomic analysisBIOMEDICAL CHROMATOGRAPHY, Issue 6 2009Atsushi Intoh Abstract Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd. [source] In situ magnetic separation for extracellular protein productionBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Tobias Käppler Abstract A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production. Biotechnol. Bioeng. 2009;102: 535,545. © 2008 Wiley Periodicals, Inc. [source] Determination of Diazepam, Temazepam and Oxazepam at the Lead Film Electrode by Adsorptive Cathodic Stripping VoltammetryELECTROANALYSIS, Issue 17-18 2010Katarzyna Tyszczuk Abstract The determination of psychoactive 1,4-benzodiazepine drugs is of relevant interest in clinical, biomedical areas. Therefore a highly sensitive and simple voltammetric method for the determination of temazepam, diazepam and oxazepam at an in situ plated lead film electrode was developed. The method was successfully applied to the determination of diazepam and temazepam in pharmaceutical formulations with minimum sample manipulation and oxazepam in human urine samples without any separation steps. The determinations of oxazepam in human urine samples were performed in a flow system. Therefore a previous extraction procedure was not necessary to separate the active compound before its determination. [source] Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonellaFEBS JOURNAL, Issue 7 2000Andreas C. Fröbius Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors. [source] Simultaneous Spectrophotometric Determination of 2-Thiouracil and 2-Mercaptobenzimidazole in Animal Tissue Using Multivariate Calibration Methods: Concerns and Rapid Methods for DetectionJOURNAL OF FOOD SCIENCE, Issue 2 2010Abolghasem Beheshti ABSTRACT:, Two multivariate calibration methods, partial least squares (PLS) and principal component regression (PCR), were applied to the spectrophotometric simultaneous determination of 2-mercaptobenzimidazole (MB) and 2-thiouracil (TU). A genetic algorithm (GA) using partial least squares was successfully utilized as a variable selection method. The concentration model was based on the absorption spectra in the range of 200 to 350 nm for 25 different mixtures of MB and TU. The calibration curve was linear across the concentration range of 1 to 10 ,g mL,1 and 1.5 to 15 ,g mL,1 for MB and TU, respectively. The values of the root mean squares error of prediction (RMSEP) were 0.3984, 0.1066, and 0.0713 for MB and 0.2010, 0.1667, and 0.1115 for TU, which were obtained using PCR, PLS, and GA-PLS, respectively. Finally, the practical applicability of the GA-PLS method was effectively evaluated by the concurrent detection of both analytes in animal tissues. It should also be mentioned that the proposed method is a simple and rapid way that requires no preliminary separation steps and can be used easily for the analysis of these compounds, especially in quality control laboratories. [source] Protein purification using chromatography: selection of type, modelling and optimization of operating conditionsJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2009J. A. Asenjo Abstract To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant , -1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (, -lactoalbumin, ovalbumin and , -lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. In order to optimize operating conditions, a cost function was built that included the effect of the different production stages, namely fermentation, purification and concentration. This cost function was also successfully used for the determination of the fraction of product to be collected (peak cutting) in chromatography. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters. Copyright © 2008 John Wiley & Sons, Ltd. [source] Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2008Lucie Korecká Abstract We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG),Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach. [source] Metabolomic analysis of Echinacea spp. by 1H nuclear magnetic resonance spectrometry and multivariate data analysis technique,PHYTOCHEMICAL ANALYSIS, Issue 1 2010Michel Frédérich Abstract Introduction , The genus Echinacea (Asteraceae) comprises about 10 species originally distributed in North America. Three species are very well known as they are used worldwide as medicinal plants: Echinacea purpurea, E. pallida, E. angustifolia. Objective , To discriminate between these three Echinacea species and E. simulata by 1H NMR-based metabolomics. Methodology , 1H NMR and multivariate analysis techniques were applied to diverse Echinacea plants including roots and aerial parts, authentic plants, commercial plants and commercial dry extracts. Results , Using the 1H NMR metabolomics, it was possible, without previous evaporation or separation steps, to obtain a metabolic fingerprint to distinguish between species. Conclusion , A clear distinction between the three pharmaceutical species was possible and some useful metabolites were identified. Copyright © 2009 John Wiley & Sons, Ltd. [source] Direct metabolic fingerprinting of commercial herbal tinctures by nuclear magnetic resonance spectroscopy and mass spectrometry,PHYTOCHEMICAL ANALYSIS, Issue 4 2009Matteo Politi Abstract Introduction Tinctures are widely used liquid pharmaceutical preparations traditionally obtained by maceration of one or more medicinal plants in ethanol,water solutions. Such a process results in the extraction of virtually hundreds of structurally diverse compounds with different polarities. Owing to the large chemical diversity of the constituents present in the herbal tinctures, the analytical tools used for the quality control of tinctures are usually optimised only for the detection of single chemical entities or specific class of compounds. Objective In order to overcome the major limitations of the current methods used for analysis of tinctures, a new methodological approach based on NMR spectroscopy and MS spectrometry has been tested with different commercial tinctures. Methodology Diffusion-edited 1H-NMR (1D DOSY) and 1H-NMR with suppression of the ethanol and water signals have been applied here for the first time to the direct analysis of commercial herbal tinctures derived from Echinacea purpurea, Hypericum perforatum, Ginkgo biloba and Valeriana officinalis. The direct injection of the tinctures in the MS detector in order to obtain the corresponding metabolic profiles was also performed. Results Using both NMR and MS methods it was possible, without evaporation or separation steps, to obtain a metabolic fingerprint able to distinguish between tinctures prepared with different plants. Batch-to-batch homogeneity, as well as degradation after the expiry date of a batch, was also investigated. Conclusion The techniques proposed here represent fast and convenient direct analyses of medicinal herbal tinctures. Copyright © 2009 John Wiley & Sons, Ltd. [source] Renewable resources , green biorefinery: separation of valuable substances from fluid,fractions by means of membrane technologyBIOFUELS, BIOPRODUCTS AND BIOREFINING, Issue 1 2009Senad Novalin Abstract The aim of this study is to emphasize the potential of membrane technologies and the specific performance-limiting borders of pressure-driven (microfiltration, ultrafiltration, nanofiltration, reverse ssmosis) as well as electro-membrane (electrodialysis, electrodialysis using bipolar membranes) techniques for the separation of valuable substances from silage press-juice obtained in green biorefineries. Depending on the product, nanofiltration can be considered a partially fractionating technique with great future potential. Electrodialysis turns out to be a suitable separation technique for removing huge amounts of salt and isolating individual valuable substances. However, residual impurities must be taken into account for subsequent separation steps. In any case, further separation processes (e.g. chromatography) must be integrated in future green biorefinery production plants. © 2008 Society of Chemical Industry and John Wiley & Sons, Ltd [source] Isolation, purification, crystallization and preliminary crystallographic studies of sagitoxin, an oligomeric cardiotoxin from the venom of Naja naja saggitiferaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2008Rafia Mir Sagitoxin, a novel cardiotoxin from the venom of Naja naja saggitifera, has been successfully isolated, purified to homogeneity and crystallized. The toxin was purified using successive separation steps on a CM-Sephadex C-50 column and a reverse-phase column. The 6.75,kDa toxin was sequenced by the Edman method using a PPSQ-21 protein sequencer. It was crystallized using the hanging-drop vapour-diffusion method. The hexagonal-shaped crystals diffracted to 3.0,Ĺ resolution and belonged to space group P64, with unit-cell parameters a = b = 111.1, c = 137.3,Ĺ, , = 120°. There are 36 molecules in the unit cell, which has an approximate solvent content of 80%. Structure determination revealed that the molecules of sagitoxin associate in a hexameric form and create a pore in the centre which has functional significance. [source] Development of a Novel Membrane Aerated Hollow-Fiber MicrobioreactorBIOTECHNOLOGY PROGRESS, Issue 2 2008Louis Villain A new challenge in biotechnological processes is the development of flexible bioprocessing platforms, allowing strain selection, facilitating scale-up and integrating separation steps. Miniaturization of such a cultivation system allows parallel use and the saving of resources but makes the supply of oxygen to the cells difficult. In this work we present a membrane aerated hollow-fiber microbioreactor (HFMBR) which consists of an acrylic glass module equipped with two different types of membrane fibers. Fibers of polyethersulfone and polyvinyldifluoride were used for substrate and oxygen supply, respectively. Cultivation of E. coli as model organism and production of His-tagged GFP were carried out in the extracapillary space of the membrane aerated HFMBR and compared with cultivations in shaking flask which are commonly used for screening experiments. The measurement of the oxygen transfer capacity and the online monitoring of the dissolved oxygen during the cultivation were performed using a fiber optic oxygen sensor. Online measurement of the optical density was also integrated to the bioreactor. Due to efficient oxygen transfer, a better cell growth than in the shaking flask experiments was achieved, while no negative influence on the GFP productivity was observed in the membrane aerated bioreactor. Thus the feasibility of a future integrated downstreaming could also be demonstrated. [source] | |||||||||||||