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Capillary Isoelectric (capillary + isoelectric)
Selected AbstractsCover Picture: Electrophoresis 13'09ELECTROPHORESIS, Issue 13 2009Article first published online: 20 JUL 200 Issue 13 is a special issue on "CE and CEC of Amino Acids, Peptides and Proteins" assembling 19 papers on various topics including fast, high efficient and high sensitive "CE and CEC techniques for quality control and purity determination of native and (bio)synthetic amino acids, peptides and proteins, for monitoring of their synthesis, isolation, chemical derivatization and enzymatic digestion and also for investigation of their interactions with other molecules. New methodologies, such as electrodialysis for sample preparation, chiral ligand-exchange CE, immunoaffinity CE, affinity capillary isoelectric focusing, combination of transient isotachophoretic preconcentration with capillary zone electophoresis (CZE) analysis, two-dimensional CE-mass spectrometry (MS) separations and advances in high-sensitive CE-laser induced fluorescence (LIF) and CE-electrochemiluminescence detection schemes, are widely presented here. The applications of CE and CEC methods include chiral analysis of amino acids, determination of low abundant amino acids, peptides and proteins in complex matrices, such as human and animal body fluids and tissue biopsies, and profiling of cell lysates and recombinant proteins, e.g. birch pollen allergen and human interleukin 7. As can be seen from several contributions, preparation of new capillary coatings suppressing the adsorption of peptides and proteins to the fused silica capillary wall in their CZE analyses and/or increasing the selectivity of their open-tubular CEC separations remains a hot topic in the area of CE and CEC developments. In addition, it is shown that through the theoretical modelling of the CZE determined effective electrophoretic mobilities of proteins, the important parameters, such as charge, hydration and shape of their molecules, can be estimated." [source] Cover Picture: Electrophoresis 2'09ELECTROPHORESIS, Issue 2 2009Article first published online: 9 FEB 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Issue no. 2 has a "Fast Track" paper on the attomole protein analysis by capillary isoelectric focusing (CIEF) with LIF detection based on a post-column sheath flow cuvette employing Chromeo P503 as a fluorogenic reagent for protein labeling before CIEF analysis. Further selected topics of issue 2 are: Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data A PDMS sheath flow cuvette for high-sensitivity LIF measurements in CE [source] Use of quasi-isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performancesELECTROPHORESIS, Issue 8 2008Martine Poitevin Abstract The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes. [source] A capillary holder for scanning detection of capillary isoelectric focusing with laser-induced fluorescenceJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2009Katsuyoshi Takahashi Abstract A holder for a 12 cm long capillary was designed for scanning LIF detection of CIEF. The polyimide coat of a fused-silica capillary has been removed, and 1.5 mm diameter flanges have been attached near both ends. The holder is fixed on the stage of a fluorescence microscope via a translational stage, and a capillary guide is directly fixed on the microscope stage. The guide has a groove and a pressure plate for the capillary to slide in. The holder has two pulling plates with slits of 1 mm to accept the capillary just inside the flanges. The slits and the groove of the guide have been aligned. The motion of the translational stage brings the pulling plate into contact with the flange at the pulled side, and slides the capillary through the guide. The other end of the capillary is free and produces no strain on the capillary. When the motion of the stage is reversed, an unstrained contact is achieved at the other end. The baseline noise from scanning was only 50% larger than that without scanning. The fluorescence-signal variation during scanning was about 4% of the total signal, which was about twice that without scanning. [source] Investigation of interaction between human hemoglobin A0 and platinum anticancer drugs by capillary isoelectric focusing with whole column imaging detectionJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2008Tibebe Lemma Abstract CIEF with whole column imaging detection (WCID) was used to investigate the interaction of platinum-based anticancer drugs, cis -platinum(II) diamine dichloride (cisplatin) and [SP-4-2-{1R-trans)]-(1,2-cyclohexanediamine- N,N,)[ethanedioata(2,)- O,O,]platinum (oxaliplatin), with human hemoglobin A0 (Hb). This technique facilitates the investigation and characterization of the formation of adducts between drugs and proteins. Cisplatin and oxaliplatin were mixed with the target protein at different concentrations (0:1, 1:1, 1:10, 1:50, and 1:100), and the reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48, and 72 h at 37°C in a water-bath. The focused Hb,drug adduct profiles were imaged by WCID. At higher drug to protein molar ratios (for both oxaliplatin and cisplatin), the results exhibit significant changes in the peak shapes and heights, which may indicate the destabilization of the protein. However, the conformational change was less evident at lower molar ratios. In addition, a major pI shift was observed for the oxaliplatin reaction mixtures (for 1:10, 1:50, and 1:100 ratios). In comparison with previously reported findings obtained by other analytical methods, conclusions were drawn about the validity of CIEF as a simple and convenient method for the investigation of protein,drug interactions. These results may provide useful information for further understanding the activity and toxicity of these chemotherapeutic drugs and improving their clinical performance. [source] Protein thermal stability and phospholipid,protein interaction investigated by capillary isoelectric focusing with whole column imaging detectionJOURNAL OF SEPARATION SCIENCE, JSS, Issue 7 2006Tao Bo Abstract CIEF with whole column imaging detection (WCID) is an attractive technique for studying protein reaction and protein,ligand interaction due to its fast separation, simple operation, and high efficiency. In this study, two interesting applications by the CIEF-WCID were developed, involving the study of protein thermal stability and phospholipid,protein interaction. Four proteins (,-lactoglobulin B, trypsin inhibitor, phosphorylase b, and trypsinogen) with different pI, and two types of phospholipids, including phosphatidylcholine (PC) and phosphatidylserine (PS), were used for this purpose. First, the altered CIEF profiles of four proteins were exhibited due to conformational changes resulting from protein denaturation induced by a high incubation temperature at 60°C. It was demonstrated that the addition of a zwitterionic phospholipid (PC) played a crucial role in the thermal stability of targeted proteins, especially for trypsin inhibitor whose thermal stability was promoted with the addition of the PC vesicles at 60°C. Second, the zwitterionic (PC) and acidic (PS) phospholipids displayed completely different interactions with the proteins. The addition of PS vesicles modified the zwitterionic phospholipids to carry negative charges, which correspondingly changed the interaction between the phospholipid and the protein. Our study demonstrates that the CIEF-WCID is a powerful approach to study protein reaction and protein,ligand interaction with high efficiency, high selectivity, and fast separation. [source] Development of a high throughput protein a well-plate purification method for monoclonal antibodiesBIOTECHNOLOGY PROGRESS, Issue 5 2009Jennifer Hopp Abstract We have developed a new high throughput method for the purification of monoclonal antibodies from harvested cell culture fluid for analytical characterization. This method uses Protein A resin in a 96 well-plate format with protein loading sufficient to perform multiple analyses per well. Resin and buffer conditions were optimized to obtain aggregate and charge variant comparability with three preparative Protein A purified monoclonal antibodies. We are able to successfully demonstrate comparability for aggregate within 0.25% based upon size-exclusion chromatography. Acidic species were found to be within 2% from the preparative purified control based upon cation-exchange chromatography, 5% based upon capillary zone electrophoresis, and 3% based upon imaged capillary isoelectric focusing. Glycan distribution was analyzed and was within 1% of the preparative purified controls. A tryptic digest was performed and all peaks in the preparative purified control were found in the first elution from the well-plate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Preclinical Manufacture of Anti-HER2 Liposome-Inserting, scFv-PEG-Lipid Conjugate.BIOTECHNOLOGY PROGRESS, Issue 1 2005Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 °C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n -butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations. [source] | ||||||||||