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mM Tris (mm + tri)
Selected AbstractsCapillary electrophoretic and computational study of the complexation of valinomycin with rubidium cationELECTROPHORESIS, Issue 5 2009Sille Ehala Abstract This study is focused on the characterization of interactions of valinomycin (Val), a macrocyclic dodecadepsipeptide antibiotic ionophore, with rubidium cation, Rb+. Capillary affinity electrophoresis was employed for the experimental evaluation of the strength of the Val,Rb+ complex. The study involved the measurement of the change of effective electrophoretic mobility of Val at increasing concentration of Rb+ cation in the BGE. From the dependence of Val effective electrophoretic mobility on the Rb+ cation concentration in the BGE (methanolic solution of 100,mM Tris, 50,mM acetic acid, 0,1,mM RbCl), the apparent binding (stability) constant (Kb) of the Val,Rb+ complex in methanol was evaluated as log,Kb=4.63±0.27. According to the quantum mechanical density functional theory calculations employed to predict the most probable structure of Val,Rb+ complex, Val is stabilized by strong non-covalent bond interactions of Rb+ with six ester carbonyl oxygen atoms so that the position of the "central" Rb+ cation in the Val cage is symmetric. [source] Speciation of arsenic compounds in fish and oyster tissues by capillary electrophoresis-inductively coupled plasma-mass spectrometryELECTROPHORESIS, Issue 7-8 2005Ching-Fen Yeh Abstract A capillary electrophoresis-inductively coupled plasma-mass spectrometric (CE-ICP-MS) method for the speciation of six arsenic compounds, namely arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid, dimethylarsinic acid, arsenobetaine and arsenocholine is described. The separation has been achieved on a 70,cm length×75,µm,ID fused-silica capillary. The electrophoretic buffer used was 15,mM Tris (pH,9.0) containing 15,mM sodium dodecyl sulfate (SDS), while the applied voltage was set at +22,kV. The arsenic species in biological tissues were extracted into 80%,v/v methanol-water mixture, put in a closed centrifuge tube and kept in a water bath, using microwaves at 80°C for 3,min. The extraction efficiencies of individual arsenic species added to the sample at 0.5,µg As/g level were between 96% and 107%, except for As(III), for which it was 89% and 77% for oyster and fish samples, respectively. The detection limits of the species studied were in the range 0.3,0.5,ng As/mL. The procedure has been applied for the speciation analysis of two reference materials, namely dogfish muscle tissue (NRCC DORM-2) and oyster tissue (NIST SRM 1566a), and two real-world samples. [source] Determination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescenceELECTROPHORESIS, Issue 18-19 2004Lijun Yu Abstract High-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantitation of intact bacteria. We demonstrate that a pathogen (Edwardsiella tarda) which causes systemic infection in commercially important fish species can be rapidly identified and determined (<10 min) after direct injection into fish fluid by CE blue light-emitting diode (LED)-induced fluorescence. SYTO 13 (488 nm/509 nm), a cell-permeable green nucleic acid stain, was used to stain the cells. Remarkably high efficiency (>1 200,000 theoretical plates/m) was achieved with this rapid and efficient CE method. It was found that proper sample vortexing (90 s) would be beneficial to disperse aggregated cells and facilitate the focusing of intact cells during electrophoresis. Ionization of the surface constituents of Edwardsiella tarda cells provided efficient surface charges for the intact cells to be separated from the EOF and damaged or lysed cells when the separation was performed in running buffer (3.94 mM Tris, 0.56 mM borate, 0.013 mM EDTA) at pH 10.5. The limit of detection (LOD) and recovery were found to be 4.2×104 cells/mL and 70.0%, respectively. This proposed CE method could become an effective tool for diagnosis and tracking of certain diseases caused by bacteria in fish species as well as in human beings. [source] Simultaneous determination of substrate and product in the process of preparation of valienamine by capillary zone electrophoresisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 13 2010Xiao-Dong Wei Abstract A simple and rapid CZE method was established for the simultaneous determination of valienamine, acarbose and validamycin A, using a 20-kV CZE with the detection wavelength of 193,nm and 50,mM phosphoric acid,20,mM Tris (pH 5.3) as a running buffer. The calibration curves of valienamine, acarbose, and validamycin A showed a good linear relationship at a concentration range of 5,1000,,g/mL. The detection limits of valienamine, acarbose, and validamycin A were 0.3, 0.6, and 0.6,,g/mL, respectively, and the average recoveries of each of the above were 99.9, 99.5, and 100.3%. The method has been successfully applied for simultaneous determination of substrate and product in the process of preparation of valienamine. [source] Enantioseparation of nuarimol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selectorJOURNAL OF SEPARATION SCIENCE, JSS, Issue 18 2008Maria Amparo Martínez-Gómez Abstract The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC-partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with HSA by CE-frontal analysis was performed. The binding parameters obtained for the first site of interaction were n1 = 0.84; K1 = 9.7 ± 0.3×103 M,1 and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 ± 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 ,M HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2,8×10,4 M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30°C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA. [source] Determination of uric acid in plasma and allantoic fluid of chicken embryos by capillary electrophoresisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 12 2007Jana Mat, ková Abstract Capillary electrophoresis with diode array detection (DAD) was used to determine uric acid (UA) in chicken plasma and the allantoic fluid of chicken embryos. Complete separation of uric and ascorbic acids was attained in less than 10 min in the optimized BGE containing 60 mM MES + 30 mM Tris + 0.001% (w/v) polybrene (pH 6.1). The limit of UA detection (0.2 mg/L) was found to be low enough for sensitive analysis of native plasma and allantoic fluid samples. Range of linearity (1,200 mg/L), repeatability for peak area (CV <4.1%) and migration time (CV <2.5%), as well as recovery of UA from biological samples (97,100%), were found to be satisfactory. The method was applied to detect the elevated UA concentrations (hyperuricemia) in chicken embryos with induced unilateral renal agenesis. CE/DAD analysis of the chicken plasma can be carried out with a relatively small volume of samples (1 ,L). [source] An efficient solubilization buffer for plant proteins focused in immobilized pH gradientsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2003Valérie Méchin Abstract The solubilization of a large array of proteins before electrophoresis itself is a very critical point for proteomic analyses. We compared the efficiency of several different solubilization buffers. From this work, we defined a very efficient solubilization buffer, including two chaotropes, two reducing agents (R2), two detergents (D2), and two kinds of carrier ampholytes in combination. This so-called R2D2 buffer (5 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, 2% N -decyl- N,N -dimethyl-3-ammonio-1-propane-sulfonate, 20 mM dithiothreitol, 5 mM Tris(2-carboxyethyl) phosphine, 0.5% carrier ampholytes 4,6.5, 0.25% carrier ampholytes 3-10) proved to be very efficient for a large range of different samples and allowed us to obtain two-dimensional gels of high resolution and quality. [source] Crystallization and preliminary X-ray crystallographic analysis of p24, a component of the potato nuclear factor PBF-2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002Darrell Desveaux The Solanum tuberosum (potato) nuclear factor PBF-2 is implicated in pathogen-induced expression of the pathogenesis-related gene PR-10a. Crystals of the DNA-binding component of PBF-2, p24, have been obtained at 277,K in 20,mM Tris,HCl pH 8.0. Recombinant protein with a His tag at its C-terminus was overexpressed in Escherichia coli in the presence and absence of selenomethionine and was purified using a combination of HiTrap affinity columns and gel-filtration chromatography. Crystals suitable for structural analysis were obtained for both native and selenomethionine-labelled proteins and yielded diffraction data at 100,K that were processed to 2.3 and 2.8,Å resolution, respectively. The p24 protein crystals belong to space group P212121, with unit-cell parameters a = 69.4,(69.1), b = 89.4,(90.5), c = 144.1,(144.3),Å. The asymmetric unit contains four protomers, giving a crystal volume per protein mass (VM) of 2.23,Å3,Da,1 and a solvent content of 45% by volume. [source] Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Zhijie Li Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid ,-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12,mg,ml,1) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100,mM Tris,HCl pH 8.0, 150,mM sodium chloride, 200,mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8,Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3,Å, , = , = 90.0, , = 124.0°. A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient VM of 2.76,Å3,Da,1 and a solvent content of 55%. [source] Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Hannes Uchtenhagen 7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50,mM ammonium sulfate, 100,mM Tris,HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275,K. The analysed crystals belonged to the rhombohedral space group P3221, with unit-cell parameters a = b = 100.1, c = 196.8,Å, and diffracted to 2.7,Å resolution. [source] Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2007Gauri Misra Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2,µl 10,mg,ml,1 protein in 50,mM Tris,HCl pH 8.0, 50,mM NaCl, 5,mM EDTA equilibrated against 500,µl reservoir solution consisting of 2.25,M ammonium formate and 0.1,M HEPES buffer pH 7.25. Diffraction data extending to 2.0,Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3,Å. The crystals are hexagonal in shape and belong to space group P6322. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (VM) of 2.1,Å3,Da,1 and a solvent content of about 36.9%. [source] Crystallization and preliminary structure analysis of the blue laccase from the ligninolytic fungus Panus tigrinusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005Marta Ferraroni The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296,K using the sitting-drop vapour-diffusion method in 22%(w/v) PEG 4000, 0.2,M CaCl2, 100,mM Tris,HCl pH 7.5. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, , = 97.7°, and contain 46% solvent. A complete native data set was collected to 1.4,Å resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes. [source] | ||||||||||