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1.
The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.  相似文献   
2.
An essential part of the modulation of protein‐binding capacity in hydrophobic interaction chromatography is the buffer‐salt system. Besides using “single” electrolytes, multicomponent electrolyte mixtures may be used as an additional tool. Both the protein solubility and the binding capacity depend on the position of a salt in the so‐called Hofmeister series. Specific interactions are observed for an individual protein‐salt combination. For salt mixtures, selectivity, recovery, and binding capacity do not behave like for the single salts that are positioned in between the two mixed components in the Hofmeister series, as the continuous correlation would suggest. Thus, finding strategies for mixed salts could potentially lead to improved capacities in hydrophobic interaction chromatography. Mixtures of ammonium sulfate, sodium citrate, sodium sulfate, sodium chloride, sodium acetate, and glycine were used to investigate the binding capacities for lysozyme and a monoclonal antibody on various hydrophobic resins. Resin capacity for two investigated proteins increases when mixtures consisting of a chaotropic and a kosmotropic salt are applied. It seems to be related to the rather basic isoelectric points of the proteins.  相似文献   
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An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC-MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC-MS/MS protein identification will be discussed.  相似文献   
5.
Proteomic methods were used to identify the levels of impurities in three commercial plasma‐derived clotting factor VIII‐von Willebrand factor (FVIII/VWF) concentrates. In all three concentrates, significant amounts of other plasma proteins were found. In Octanate and Haemoctin, two concentrates developed in the 1990s, the major impurities identified were inter‐α inhibitor proteins, fibrinogen and fibronectin. These two concentrates were also found to contain additional components such as clotting factor II (prothrombin) that are known activators of FVIII. In Wilate, a recently developed FVIII/VWF concentrate, the amount of these impurities was significantly reduced. Batch‐to‐batch variations and differences between three investigated products were detected using iTRAQ, an isotope labeling technique for comparative MS, demonstrating the potential value of this technique for quality control analysis. The importance of thorough proteomic investigations of therapeutic FVIII/VWF preparations from human plasma is also discussed.  相似文献   
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Convective interaction media (CIM) monoliths provide a stationary phase with a high binding capacity for large molecules and are capable of high flow rates at a very low pressure drop. Used as anion- and cation-exchangers or with affinity ligands such as antibodies, these columns have the potential for processing large volumes of complex biological mixtures within a short time. In the present report, monoclonal antibodies against several rat liver plasma membrane proteins were bound and cross-linked to protein A or protein G CIM affinity columns with a bed volume of only 60 microL. Antigens recognized by bound antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step from either total plasma membrane extracts or subfractions isolated using anion-exchange CIM disk-shaped columns. The isolated antigens and co-eluting proteins were subsequently identified by immunoblot or by LC-MS/MS.  相似文献   
8.
The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.  相似文献   
9.
An overview is given on the application of proteomic technology in the monitoring of different steps during the production of therapeutic proteins from human plasma. Recent advances in this technology enable the use of proteomics as an advantageous tool for the validation of already existing processes, the development and fine tuning of new production steps, the characterization and quality control of final products, the detection of both harmful impurities and modifications of the therapeutic protein and the auditing of batch-to-batch variations. Further, use of proteomics for preclinical testing of new products, which can be either recombinant or plasma-derived, is also discussed.  相似文献   
10.
We analyze the dependence of the Gross Domestic Product (GDP) per capita growth rates on changes in the Corruption Perceptions Index (CPI). For the period 1999–2004 for all countries in the world, we find on average that an increase of CPI by one unit leads to an increase of the annual GDP per capita growth rate by 1.7%. By regressing only the European countries with transition economies, we find that an increase of CPI by one unit generates an increase of the annual GDP per capita growth rate by 2.4%. We also analyze the relation between foreign direct investments received by different countries and CPI, and we find a statistically significant power-law functional dependence between foreign direct investment per capita and the country corruption level measured by the CPI. We introduce a new measure to quantify the relative corruption between countries based on their respective wealth as measured by GDP per capita.  相似文献   
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