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Buffer Composition (buffer + composition)
Selected AbstractsHigh-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] Chiral CE of aromatic amino acids by ligand-exchange with zinc(II),L -lysine complexELECTROPHORESIS, Issue 15 2007Li Qi Abstract A novel method of chiral ligand-exchange CE was developed with either L - or D -lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5,mM ammonium acetate, 100,mM boric acid, 3,mM ZnSO4·7H2O and 6,mM L -Lys at pH,7.6, unlabeled D,L -tryptophan, D,L -phenylalanine, and D,L -tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2,4,mM and running buffer pH,7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L -Lys to D -Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L -isomers eluted before or after D -isomers in excessive D - or L -Lys, respectively. [source] Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretinELECTROPHORESIS, Issue 14 2003Klaus Altland Abstract Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70,80°C in neutral to mild alkaline buffers or at 37°C and slightly acidic pH (6,7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis. [source] Cytochrome b559 content in isolated photosystem II reaction center preparationsFEBS JOURNAL, Issue 10 2003Inmaculada Yruela The cytochrome b559 content was examined in five types of isolated photosystem II D1-D2-cytochrome b559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer composition and detergent concentration. Two different types of cytochrome b559 assays were performed. The absolute heme content in each preparation was obtained using the oxidized-minus-reduced difference extinction coefficient of cytochrome b559 at 559 nm. The relative amount of D1 and cytochrome b559,-subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the cytochrome b559 heme content in photosystem II reaction center complexes can vary with the isolation procedure, but the variation of the cytochrome b559,-subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their ,-subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 cytochrome b559,-subunit per 1.0 D1 polypeptide in photosystem II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes. [source] Structural characterization of a neurotoxic threonine-rich peptide corresponding to the human prion protein ,2-helical 180,195 segment, and comparison with full-length ,2-helix-derived peptides,JOURNAL OF PEPTIDE SCIENCE, Issue 10 2008Luisa Ronga Abstract The 173,195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ,spots' of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation-prone conformations. Here, we report CD and NMR studies on the ,2-helix-derived peptide of maximal length (hPrP[180,195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other ,2-helix-derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C -terminal sequence of the PrPC full-length ,2-helix and includes the highly conserved threonine-rich 188,195 segment. At neutral pH, its conformation is dominated by ,-type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of ,-helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173,179 segment, as occurring in wild-type and mutant peptides corresponding to the full-length ,2-helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180,195 parental region in hPrPC makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full-length ,2-helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Electrogenerated chemiluminescence of luminol for oxidase-based fibre-optic biosensorsLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 2 2001Christophe A. Marquette Abstract The luminol electrochemiluminescence has been exploited for the development of several fibre-optic biosensors allowing the detection of hydrogen peroxide and of substrates of H2O2 -producing oxidases. Electro-optical flow injection analysis of glucose, lactate, cholesterol and choline are thus described. To perform the experiments, a glassy carbon electrode was polarized at a fixed potential. Luminol was then electrochemically oxidized and could react in the presence of hydrogen peroxide to produce light. Several parameters had to be optimized to obtain reliable optical biosensors. An optimum applied potential of +425 mV between the glassy carbon electrode and the platinum pseudo-reference electrode was determined, allowing the best signal: noise ratio to be obtained. It was also necessary to optimize the experimental conditions for the immobilization of the different oxidases involved (preactivated membranes, chemically activated collagen membranes, photopolymerized matrix). For each biosensor developed, the optimum reaction conditions have been studied: buffer composition, pH, temperature, flow rate and luminol concentration. Under optimal conditions, the detection limits (S/N,=,3) were 30,pmol, 60,pmol, 0.6,nmol and 10,pmol for lactate, glucose, cholesterol and choline, respectively. The miniaturization of electrochemiluminescence-based biosensors has been realized using screen-printed electrodes instead of a glassy carbon macroelectrode, with choline oxidase as a model H2O2 -generating oxidase. Copyright © 2001 John Wiley & Sons, Ltd. [source] A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopyBIOTECHNOLOGY JOURNAL, Issue 9-10 2008Patrick Garidel Dr. Abstract Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed. [source] Effect of antibody solution conditions on filter performance for virus removal filter PlanovaÔ 20NBIOTECHNOLOGY PROGRESS, Issue 4 2010Tomoko Hongo-Hirasaki Abstract We investigated the effect of antibody solution conditions (ionic strength, pH, IgG concentration, buffer composition, and aggregate level (dimer content)) on filter performance for a virus removal filtration process using the PlanovaÔ 20N, a virus removal filter. Ionic strength and pH affected the filter flux. A consistent high flux was maintained at an ionic strength greater than 10 mM and at pH 4,8 under a typical buffer composition (sodium chloride, citrate, acetate, and phosphate). Optimum IgG concentration was 10,20 mg/mL allowing for high throughput (kg/m2 of IgG). Dimer content negligibly affected the flux level. Under high throughput conditions, virus spiking did not affect flux whereas a parvovirus logarithmic reduction value greater than 5 was maintained. From the results of zeta potential analyses for IgG and the membrane, we considered that electrostatic interactions between antibodies and the membrane affect filter performance (flux level and throughput). These results indicate that the PlanovaÔ 20N filter is applicable for a wide range of solution conditions typically used in antibody processing. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | |||||||||||||