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Protein Separation (protein + separation)

Distribution by Scientific Domains
Life Sciences46%
Chemistry44%
Polymers and Materials Science4%
2 Other Domains6%

Selected Abstracts

Endochitinase activity in the apoplastic fluid of Phellinus weirii -infected Douglas-fir and its association with over wintering and antifreeze activity

FOREST PATHOLOGY, Issue 5 2003
A. Zamani
Summary Extracellular proteins were extracted from Phellinus weirii infected Douglas-fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase-like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two-dimensional gel electrophoresis (2-D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27,30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii -infected winter Douglas-fir needles showed anti-freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response. Résumé Les protéines extra-cellulaires ont été extraites des racines et aiguilles de douglas (Pseudotsuga menziesii var menziesii) infectés par Phellinus weirii (Murr.) Gilbn., pour étudier l'activité endochitinase. Les chitinases ont été associées aux réactions de défense des plantes contre les attaques fongiques parce-qu'elles hydrolysent la chitine, un composant de la paroi des cellules fongiques. La séparation des protéines, réalisée par électrophorèse en gel de polyacrylamide avec sodium dodecyl sulfate (SDS-PAGE), suivie par une analyse par Western immunoblot en utilisant un anticorps polyclonal spécifique d'une protéine de type endochitinase (ECP), a permis la détection de 3 polypeptides de taille comprise entre 27 et 30 kDa. Une électrophorèse sur gel en 2-dimensions (2-D PAGE) suivie par une analyse par Western immunoblot a révélé que le fluide apoplastique contient de multiples isoformes d'ECP avec des pI dans une gamme de 5.3 à 5.8 et des masses moléculaires de 27 à 30 kDa. L'activité chitinase dans les aiguilles et tissus racinaires a été mesurée par spectrophotométrie par une méthode colorimétrique. Une technique d'overlay utilisant de la chitine glycol comme substrat de l'endochitinase a été appliquée pour confirmer que l'anticorps ECP avait détecté une protéine active du point de vue enzymatique. Le fluide apoplastique d'aiguilles récoltées en hiver sur des douglas infectés par P. weirii a montré une activité antigel et l'analyse saisonnière des tissus foliaires a montré une certaine accumulation d'ECP pendant l'hiver. L'ECP est répartie de façon systémique dans l'ensemble de l'arbre. Les niveaux accrus d'activité endochitinase dans la zone infectée par P. weirii suggère un rôle physiologique de l'ECP dans les réactions de défense de la plante. Zusammenfassung Aus Wurzeln und Nadeln von mit Phellinus weirii infizierten Douglasien (Pseudotsuga menziesii var. menziesii) wurden extrazelluläre Proteine extrahiert, um die Endochitinase-Aktivität zu bestimmen. Chitinasen werden mit der pflanzlichen Abwehrreaktion auf Pilzinfektionen in Verbindung gebracht, da sie Chitin, eine Strukturkomponente der pilzlichen Zellwand, hydrolysieren. Die Proteine wurden mit Natrium-Dodecyl-Sulfat-Polyacrylamid-Gelelektrophorese (SDS-PAGE) getrennt, gefolgt von einer Western Immunoblot-Analyse mit einem gegen ein Endochitinase-ähnliches Protein (ECP) spezifischen polyklonalen Antikörper. Hiermit liessen sich bis zu drei Polypeptide zwischen 27-30 kDa nachweisen. Eine zweidimensionale Gelelektrophorese (2-D PAGE) mit anschliessender Western Immunoblot-Analyse ergab, dass die Apoplastenflüssigkeit multiple ECP-Isoformen enthielt (mit pIs von 5,3 bis 5,8 und Molekularmassen von 27 bis 30 kDa). Die Chitinase-Aktivität wurde auch im Nadel- und Wurzelgewebe spektrophotometrisch mit einer Farbreaktion gemessen. Um sicher zu stellen, dass der ECP-Antikörper ein enzymatisch aktives Protein nachwies, wurde eine Gel-Overlay-Methode verwendet, mit Glycolchitin als Substrat für die Endochitinase. Die Apoplastenflüssigkeit der Nadeln von mit P. weirii infizierten Douglasien zeigte in Winterzustand eine Antifrost-Aktivität, ihre Analyse während des gesamten Jahres ergab aber keine Hinweise auf eine ECP-Anreicherung während der Wintermonate. ECP war systemisch im gesamten Baum enthalten. Die erhöhte Endochitinase-Aktivität in Bereichen mit P. weirii -Infektion lässt auf eine physiologische Rolle von ECP in der Pflanzenabwehr schliessen. [source]

Allelic variants of granule-bound starch synthase proteins in European bread wheat varieties

PLANT BREEDING, Issue 4 2000
C. Marcoz-Ragot
Abstract The composition of 324 European wheat cultivars were analysed at the three granule-bound starch synthase (GBSS I) loci. Protein separation was first made by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE. A specific two-dimensional (2D) electrophoresis (immobilized pH gradient × SDS-PAGE) using an Immobiline dry strip in the first dimension was developed to resolve the GBSS I proteins more clearly and to confirm some results. Very low polymorphism was found. Among the 324 cultivars analysed, only one carried a Wx-A1 null allele (Wx-A1b) and none was found to have the Wx-2D null allele. As described in the literature the Wx-B1 locus was more polymorphic and the null allele was encountered in 11 cultivars. The use of 2D electrophoresis allowed us to find another type of variant which presented as having thicker band with same mobility as the Wx-D1 protein in SDS-PAGE. Twelve per cent of the cultivars analysed presented this band and could have been previously mistaken for cultivars carrying the Wx-B1 null allele. Indeed this band probably corresponded to the Wx-B1, or Wx-B1e allele overlapping with the Wx-D1a allele in SDS-PAGE. [source]

Protein separations using polyelectrolyte multilayer coatings with molecular micelles in open tubular capillary electrochromatography

ELECTROPHORESIS, Issue 4 2008
Candace A. Luces
Abstract Novel polyelectrolyte multilayer (PEM) coatings for enhanced protein separations in open tubular CEC (OT-CEC) are reported. Use of four cationic polymers (poly- L -lysine, poly- L -ornithine, poly- L -lysine-serine, and poly- L -glutamic acid-lysine), and three anionic molecular micelles, sodium poly(N -undecanoyl- L -leucyl-alaninate) (poly- L -SULA), sodium poly(N -undecanoyl- L -leucyl-valinate) (poly- L -SULV), and sodium poly(undecylenic sulfate) (poly-SUS) were investigated in PEM coatings for protein separations. The simultaneous effects of cationic polymer concentration, number of bilayers, temperature, applied voltage, and pH of the BGE on the separation of four basic proteins (,-chymotrypsinogen A, lysozyme, ribonuclease A, and cytochrome c) were analyzed using a Box Behnken experimental design. The influence of NaCl on the run-to-run reproducibility was investigated for PEM coatings containing each cationic polymer. All coatings exhibited excellent reproducibilities with a %RSD of the EOF less than 1% in the presence of NaCl. Optimal conditions were dependent on both the cationic and anionic polymers used in the PEM coatings. Poly- L -glutamic acid-lysine produced the highest resolution and longest migration time. The use of molecular micelles to form PEM coatings resulted in better separations than single cationic coatings. Chiral poly- L -SULA and poly- L -SULV resulted in higher protein resolutions as compared to the achiral, poly-SUS. Furthermore, the use of poly- L -SULV reversed the elution order of lysozyme and cytochrome c when compared to poly- L -SULA and poly-SUS. [source]

Oscillatory transverse electric field enhances protein resolution and capacity of size-exclusion chromatography

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 5 2006
Guo-Min Tan
Abstract Protein separations by a novel size-exclusion electrochromatography (SEEC) are presented. The present SEEC, denoted as pSEEC, was established with an oscillatory low-voltage electric field perpendicular to the mobile-phase streamline. Retention experiments with different proteins indicated that the influence of electric field strength on the partition coefficient is different for different proteins as well as for the same protein under different mobile-phase conditions. These results of protein retention led to the experimental design of protein separations with binary mixtures of BSA and immunoglobulin G (IgG), myoglobin (Myo) and lysozyme (Lys), as well as ovalbumin (Oval) and Myo. The separation results for the binary protein systems sufficiently exhibited the applicability of the pSEEC for various separations in terms of their molecular weights (MWs) as well as pIs. For example, it was possible to separate the gel-excluded proteins (BSA/IgG) as well as gel-permeable and similar-molecular-weight proteins (Myo/Lys) by the pSEEC. Moreover, in the cases of Oval/Myo, which could be partially separated by size-exclusion chromatography, the use of the pSEEC greatly improved the resolution and the separation became possible at high sample loading. The results indicate that the pSEEC technology is promising for preparative protein separations. [source]

Cytotoxicity and Cell Cycle Effects of Bare and Poly(vinyl alcohol)-Coated Iron Oxide Nanoparticles in Mouse Fibroblasts

ADVANCED ENGINEERING MATERIALS, Issue 12 2009
Morteza Mahmoudi
Super-paramagnetic iron oxide nanoparticles (SPIONs) are recognized as powerful biocompatible materials for use in various biomedical applications, such as drug delivery, magnetic-resonance imaging, cell/protein separation, hyperthermia and transfection. This study investigates the impact of high concentrations of SPIONs on cytotoxicity and cell-cycle effects. The interactions of surface-saturated (via interactions with cell medium) bare SPIONs and those coated with poly(vinyl alcohol) (PVA) with adhesive mouse fibroblast cells (L929) are investigated using an MTT assay. The two SPION formulations are synthesized using a co-precipitation method. The bare and coated magnetic nanoparticles with passivated surfaces both result in changes in cell morphology, possibly due to clustering through their magnetostatic effect. At concentrations ranging up to 80,×,10,3,M, cells exposed to the PVA-coated nanoparticles demonstrate high cell viability without necrosis and apoptosis. In contrast, significant apoptosis is observed in cells exposed to bare SPIONs at a concentration of 80,×,10,3,M. Nanoparticle exposure (20,80,×,10,3,M) leads to variations in both apoptosis and cell cycle, possibly due to irreversible DNA damage and repair of oxidative DNA lesions, respectively. Additionally, the formation of vacuoles within the cells and granular cells indicates autophagy cell death rather than either apoptosis or necrosis. [source]

Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresis

ELECTROPHORESIS, Issue 17 2010
Sílvia Barrabés
Abstract 2-DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, ,1-antitrypsin and ribonuclease 1), which N -glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule. [source]

Cationic and anionic lipid-based nanoparticles in CEC for protein separation

ELECTROPHORESIS, Issue 11 2010
Christian Nilsson
Abstract The development of new separation techniques is an important task in protein science. Herein, we describe how anionic and cationic lipid-based liquid crystalline nanoparticles can be used for protein separation. The potential of the suggested separation methods is demonstrated on green fluorescent protein (GFP) samples for future use on more complex samples. Three different CEC-LIF approaches for protein separation are described. (i) GFP and GFP N212Y, which are equally charged, were separated with high resolution by using anionic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (ii) High efficiency (800,000 plates/m) and peak capacity were demonstrated separating GFP samples from Escherichia coli with cationic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (iii) Three single amino-acid-substituted GFP variants were separated with high resolution using an approach based on a physical attached double-layer coating of cationic and anionic nanoparticles combined with anionic lipid nanoparticles suspended in the electrolyte. The soft and porous lipid-based nanoparticles were synthesized by a one-step procedure based on the self-assembly of lipids, and were biocompatible with a large surface-to-volume ratio. The methodology is still under development and the optimization of the nanoparticle chemistry and separation conditions can further improve the separation system. In contrast to conventional LC, a new interaction phase is introduced for every analysis, which minimizes carry-over and time-consuming column regeneration. [source]

Synthesis of poly(N, N -dimethylacrylamide)- block -poly(ethylene oxide)- block -poly(N, N -dimethylacrylamide) and its application for separation of proteins by capillary zone electrophoresis

ELECTROPHORESIS, Issue 10 2010
Jing Xu
Abstract A series of well-defined triblock copolymers, poly(N, N -dimethylacrylamide)- block -poly(ethylene oxide)- block -poly(N, N -dimethylacrylamide) (PDMA- b -PEO- b -PDMA) synthesized by atom transfer radical polymerization, were used as physical coatings for protein separation. A comparative study of EOF showed that the triblock copolymer presented good capillary coating ability and EOF efficient suppression. The effects of the Mr of PDMA block in PDMA- b -PEO- b -PDMA triblock copolymer and buffer pH on the separation of basic protein for CE were investigated. Moreover, the influence of the copolymer structure on separation of basic protein was studied by comparing the performance of PDMA- b -PEO- b -PDMA triblock copolymer with PEO- b -PDMA diblock copolymer. Furthermore, the triblock copolymer coating showed higher separation efficiency and better migration time repeatability than fused-silica capillary when used in protein mixture separation and milk powder samples separation, respectively. The results demonstrated that the triblock copolymer coatings would have a wide application in the field of protein separation. [source]

Cover Picture: Electrophoresis 20'2009

ELECTROPHORESIS, Issue 20 2009
Article first published online: 27 OCT 200
Issue no. 20 is a regular issue with an Emphasis on "Fundamentals and Methodologies". The bulk of this issue (13 articles) is on fundamentals and methodologies covering various topics, e.g. EOF, affinity CE, structural analysis of glycosphingolipids by CE-ESI-MS, on-line concentration, monolithic columns, etc. The other 6 articles are on protein separation and proteomics. Selected articles are: Micropump based on electroosmosis of the second kind ((10.1002/elps.200900271)) A splicing model-based DNA computing approach on microfluidic chip ((10.1002/elps.200900323)) Proteomic Characterization of Plasma-derived Clotting Factor VIII , von Willebrand Factor Concentrates ((10.1002/elps.200900270)) [source]

Co-electroosmotic capillary electrophoresis of basic proteins with 1-alkyl-3-methylimidazolium tetrafluoroborate ionic liquids as non-covalent coating agents of the fused-silica capillary and additives of the electrolyte solution

ELECTROPHORESIS, Issue 11 2009
Danilo Corradini
Abstract The paper reports the results of a study carried out to evaluate the use of three 1-alkyl-3-methylimidazolium-based ionic liquids as non-covalent coating agents for bare fused-silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co-EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co-electroosmotic CE is obtained with the 1-butyl-3-methylimidazolium tetrafluoroborate coated capillary and 100,mM acetate buffer (pH 4.0) containing 4.4,mM 1-butyl-3-methylimidazolium tetrafluoroborate as the BGE. [source]

Two-dimensional protein separation in microfluidic devices

ELECTROPHORESIS, Issue 5 2009
Hong Chen
Abstract Proteomics is emerging as an important tool in modern drug discoveries and medical diagnostics. One of the techniques used in proteomics studies is 2-DE. The process of the conventional 2-DE is time-consuming and it has substandard reproducibility. Many efforts have been made to address the limitations, with an aim for fast separation and high resolution. In this paper, we reviewed the work on achieving 2-DE in microfluidic devices, including individual dimension in one channel, two dimensions in two intersected channels, and 2-D separation in a large number of channels. We also discussed the need for integrating microvalves within 2-DE devices to prevent different separation media from contaminating with each other. Although more efforts are required to match the performance of conventional 2-DE in a slab gel, microfluidics-based 2-D separation has a potential to become an alternative in the future. [source]

A more practical strategy for interfacing microscale solution phase IEF with commercially available very narrow range IPG strips for optimal protein separation

ELECTROPHORESIS, Issue 12 2007
Article first published online: 18 JUN 200
This article has been removed from Wiley InterScience prior to final publication at the request of the authors. [source]

SDS-CGE of proteins in microchannels made of SU-8 films

ELECTROPHORESIS, Issue 18 2006
Maria Agirregabiria
Abstract This work describes the SDS-CGE of proteins carried out in microchannels made of the negative photoresist EPON SU-8. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology allows the monolithic integration of the electrodes in the device. A high wafer fabrication yield and mass production compatibility guarantees low costs and high reliability. A poly(methyl methacrylate) (PMMA) packaging allows an easy setup and replacement of the device for electrophoresis experiments. In addition, the wire-bonding step is avoided. The electrophoretic mobilities of four proteins have been measured in microchannels filled with polyacrylamide. Different pore sizes have been tested obtaining their Ferguson plots. Finally, a separation of two proteins (20 and 36,kDa) has been carried out confirming that this novel device is suitable for protein separation. A resolution of 2.75 is obtained. This is the first time that this SU-8 microfluidic technology has been validated for SDS-CGE of proteins. This technology offers better separation performance than glass channels, at lower costs and with an easy packaging procedure. [source]

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

ELECTROPHORESIS, Issue 19 2005
Wibke Hellmich
Abstract Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488,nm) as well as in the deep UV (266,nm) spectral range for label-free, native protein detection. Minute concentrations of 100,fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future. [source]

Application of dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium in wall modification for capillary electrophoresis separation of proteins

ELECTROPHORESIS, Issue 3 2005
Wei Wei
Abstract A zwitterionic surfactant, dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium (C12H25N+(CH3)2CH2CHOHCH2SO3,), named dodecyl sulfobetaine (DSB), was used as a novel modifier to coat dynamically capillary walls for capillary electrophoresis separation of basic proteins. The DSB coating suppressed the electroosmotic flow (EOF) in the pH range of 3,12. At high DSB concentration, the EOF was suppressed by more than 8.8,times. The DSB coating also prevented successfully the adsorption of cationic proteins on the capillary wall. Anions, such as Cl,, Br,, I,, SO42,, CO32,, and ClO4,, could be used as running buffer modifiers to adjust the EOF for better separation of analytes. Using this dynamically coated capillary, a mixture of eight inorganic anions achieved complete separation within 4.2,min with the efficiencies from 24,000 to 1,310,000,plates/m. In the presence of ClO4, as EOF adjustor, the separation of a mixture containing four basic proteins (lysozyme, cytochrome c, ,-chymotrypsinogen,A, and myoglobin) yielded efficiencies of 204,000,896,000,plates/m and recoveries of 88%,98%. Migration time reproducibility of these proteins was less than 0.5% relative standard deviation (RSD) from run to run and less than 3.1% RSD from day to day, showing promising application of this novel modifier in protein separation. [source]

Comprehensive proteome analysis by chromatographic protein prefractionation

ELECTROPHORESIS, Issue 7-8 2004
Pierre Lescuyer
Abstract Protein copy number is distributed from 7 to 8 orders of magnitude in cells and probably up to 12 orders of magnitude in plasma. Classical silver-stained two-dimensional electrophoresis (2-DE) can only display up to four orders of magnitude. This is a major drawback since it is assumed that most of the regulatory proteins are low-abundance gene products. It is thus clear that the separation of low copy number proteins in amounts sufficient for postseparation analysis is an important issue in proteome studies to complete the comprehensive description of the proteome of any given cell type. The visualization of a polypeptide on a 2-DE gel will depend on the copy number, on the quantity loaded onto the gel and on the method of detection. As the amount of protein that can be loaded onto a gel is limited, one efficient solution is to fractionate the sample prior to 2-DE analysis. Several approaches exist including subcellular fractionation, affinity purification and chromatographic and electrophoretic protein prefractionation. The chromatographic step adds a new dimension in the protein separation using specific protein properties. It allows proteins to be adsorbed to a surface and eluted differentially under certain conditions. This review article presents studies combining chromatography-based methods to 2-DE analysis and draws general conclusions on this strategy. [source]

EFFECT OF SALTS AND POLYETHYLENE GLYCOLS ON THE PARTITIONING AND RECOVERY OF TRYPSIN FROM HYBRID CATFISH VISCERA IN AQUEOUS TWO-PHASE SYSTEMS

JOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010
SAPPASITH KLOMKLAO
ABSTRACT The partitioning behavior of trypsin from hybrid catfish viscera in aqueous two-phase systems (ATPS) was studied. Factors such as polyethylene glycol (PEG) molecular mass and concentration, as well as types and concentration of salts, affected protein separation. Trypsin partitioned mainly in the top PEG-rich phase. ATPS formed by PEG of molecular weight 4,000 (20%, w/w) and NaH2PO4 (20%, w/w) showed the best capability for trypsin purification from hybrid catfish viscera. Under such conditions, the highest specific activity (30.05 units/µg protein) and purification (27.3-fold), were obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the enzyme after ATPS separation was near homogeneity and based on the activity staining, the band intensity of enzyme in ATPS fraction increased, indicating the greater specific activity of the viscera extract. The partitioned enzyme displayed optimal activity at pH 9.0 and 50C, respectively. The enzyme was stable up to 40C and within the pH range of 8,12. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration. PRACTICAL APPLICATIONS This paper describes the separation and recovery of trypsin from hybrid catfish viscera in ATPS and its properties. ATPS provides an efficient and attractive method for partitioning and recovery of trypsin from hybrid catfish viscera. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Therefore, it is expected that like other trypsins, trypsin after ATPS separation from hybrid catfish viscera could be useful in the biomedical, food and beverage industries. [source]

Exploration of ionic modification in dual-layer hollow fiber membranes for long-term high-performance protein separation

AICHE JOURNAL, Issue 2 2009
Yi Li
Abstract Two types of ionic modification approaches (i.e., sulfonation and triethylamination) were applied with the aid of dual-layer hollow fiber technology in this work to fine tune the pore size and pore size distribution, introduce the electrostatic interaction, and reduce membrane fouling for long-term high-performance protein separation. A binary protein mixture comprising bovine serum albumin (BSA) and hemoglobin (Hb) was separated in this work. The sulfonated fiber exhibits an improved BSA/Hb separation factor at pH = 6.8 compared with as-spun fibers but at the expense of BSA sieving coefficient. On the other hand, the triethylaminated fiber reveals the best and most durable separation performance at pH = 4.8. Its BSA/Hb separation factor is maintained above 80 for 4 days and maximum BSA sieving coefficient reaches 33%. Therefore, this study documents that an intelligent combination of both size-exclusion and electrostatic interaction can synergistically enhance protein separation performance in both purity and concentration. © 2008 American Institute of Chemical Engineers AIChE J, 2009 [source]

Nonlinear modeling of protein separation in a preparative-scale dynamic field gradient focusing instrument

AICHE JOURNAL, Issue 1 2009
Noah I. Tracy
Abstract Dynamic field gradient focusing (DFGF) uses an electric field gradient opposed by a counter-flow of buffer to separate milligrams of proteins according to their electrophoretic mobilities. A nonlinear model of protein separation in a preparative-scale DFGF device was developed to aid in refining the instrument's design and finding optimal run conditions prior to performing experiments. The model predicted the focal points of bovine serum albumin (BSA), and bovine hemoglobin (Hb) to within the 95% confidence intervals about the means of the experimental values. The resolution between the proteins in the model was 2.08, which was 3% less than the lower limit of the 95% confidence interval about the experimental value. The model predicted 67% more dispersion than was present in the experimental device, which made the simulated BSA peak 22% wider than the experimentally measured width. © 2008 American Institute of Chemical Engineers AIChE J, 2009 [source]

Performance of wide-pore monolithic silica column in protein separation

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 15-16 2009
Hironobu Morisaka
Abstract A monolithic wide-pore silica column was newly prepared for protein separation. The wide distribution of the pore sizes of monolithic columns was evaluated by mercury porosimetry. This column, as well as the conventional monolithic column, shows high permeability in the chromatographic separation of low-molecular-sized substances. In higher-molecular-sized protein separation, the wide-pore monolithic silica column shows better performance than that of the conventional monolithic column. Under optimized conditions, five different proteins , ribonuclease A, albumin, aldolase, catalase, and ferritin , were baseline-separated within 3 min, which is faster than that using the particle-packed columns. In addition, the monolithic wide-pore silica column could also be prepared in fused silica capillary (600 mm long, 0.2 mm i.d.) for highly efficient protein separation. The peak capacity of the wide-pore monolithic silica capillary column is estimated to be approximately 300 in the case of protein separation, which is a characteristic performance. [source]

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 13 2008
Rosa Cabrera
Abstract Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on ImmobilineTM strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock. [source]

Optimizing protein extraction from plant tissues for enhanced proteomics analysis

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 11 2008
Wei Wang
Abstract Plant tissues usually contain high levels of proteases and secondary metabolites that severely interfere with protein extraction, separation, and identification. Preparation of high-quality protein samples from plant tissues for proteomic analysis represents a great challenge. This article briefly describes the critical points in protein separation, especially secondary metabolites in plant tissues, and removal strategy. It provides an updated overview of three total protein extraction methods and their applications in proteomic analysis of various recalcitrant tissues. [source]

Performance of hollow-fiber flow field-flow fractionation in protein separation

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 16 2005
Ilyong Park
Abstract Since hollow-fiber flow field-flow fractionation (HF FlFFF) utilizes a cylindrical channel made of a hollow-fiber membrane, which is inexpensive and simple in channel assembly and thus disposable, interests are increasing as a potential separation device in cells, proteins, and macromolecules. In this study, performance of HF FlFFF of proteins is described by examining the influence of flow rate conditions and length of fiber (polyacrylonitrile or PAN in this work) on sample recovery as well as experimental plate heights. The interfiber reproducibility in terms of separation time and recovery was also studied. Experiments showed that sample recovery was consistent regardless of the length of fiber when the effective field strength (equivalent to the mean flow velocity at the fiber wall) and the channel void time were adjusted to be equivalent for channels of various fiber lengths. This supported that the majority of sample loss in HF FlFFF separation of apoferritin and their aggregates may occur before the migration process. It is finally demonstrated that HF FlFFF can be applied for characterizing the reduction in Stokes' size of low density lipoproteins from blood plasma samples obtained from patients having coronary artery disease and from healthy donors. [source]

Dye-affinity hollow-fibres and their lysozyme adsorption,desorption characteristics

POLYMER INTERNATIONAL, Issue 10 2001
Serap, enel
Abstract Dye-affinity adsorption is increasingly used for protein separation. Hollow-fibres have advantages as adsorbents in comparison to conventional bead supports because they are not compressible and can eliminate internal diffusion limitations. The aim of this study was to explore in detail the performance of polyamide hollow-fibres to which Reactive Green HE-4BD was attached for adsorption of lysozyme. The hollow-fibre was characterized by scanning electron microscopy. These dye-carrying hollow-fibres (26.3,µmol,g,1) were used in the lysozyme adsorption,elution studies. The effect of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye-attached hollow-fibres was studied in a batch system. The non-specific adsorption of lysozyme on the polyamide hollow-fibres was 1.8,mg,g,1. Reactive Green HE-4BD attachment significantly increased the lysozyme adsorption up to 41.1,mg,g,1. Langmuir adsorption model was found to be applicable in interpreting lead adsorption by Reactive Green HE-4BD attached hollow fibres. Significant amount of the adsorbed lysozyme (up to 95%) was eluted in 1,h in the elution medium containing 1.0,M NaSCN at pH 8.0. In order to determine the effects of adsorption conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We concluded that polyamide dye-affinity hollow-fibres can be applied for lysozyme adsorption without causing any significant conformational changes. Repeated adsorption,elution processes showed that these dye-attached hollow-fibres are suitable for lysozyme adsorption. © 2001 Society of Chemical Industry [source]

The 1st European Summer School on ,Proteomic Basics' , The Students View 12,18 August, 2007 Kloster Neustift, Brixen/Bressanone, South Tyrol, Italy

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008
Emily S. Collins Dr.
Abstract Fifty postgraduate and postdoctoral delegates from all over Europe attended the week-long ,1st European Summer School on Proteomic Basics' in Kloster Neustift in the Italian South Tyrol in August 2007. Invited proteomics experts gave tutorial lectures on Proteomics techniques with an emphasis on sample preparation, protein separation and purification in the first of an annual series of Proteomics Summer Schools funded by the EU and the Volkswagen Stiftung. [source]

Applications of field-flow fractionation in proteomics: Presence and future

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2007
Josef Chmelik RNDr.Article first published online: 19 JUL 200
Abstract Field-flow fractionation (FFF) represents a group of elution separation methods where external force fields act perpendicularly on analytes in a carrier liquid flows with nonuniform velocity profiles. It is an elution separation method that enables to separate analytes in relatively short times and collect fractions for further characterization or for investigation of their properties. Other advantages of FFF are small consumption of samples and gentle experimental conditions. These make FFF uniquely qualified for separation and purification of biological samples. The most promising are applications of different variants of flow FFF utilizing a cross flow through membrane channel walls to separate proteins. The separation is based on differences in protein diffusion coefficients, which allows calculating the size of macromolecules. Other FFF techniques (e.g., electrical, isoelectric, and sedimentation FFF) were also used for separation of biomolecules. FFF appears to be not only promising rapid technique for protein separation but it offers some other advantages in sample preparation, especially, focusing (hyperlayer) FFF techniques that enable preparation of homogeneous fractions of cells. Selected applications of FFF to protein analysis are described and future trends in application of FFF to proteomics are discussed. [source]

Immunoaffinity separation of plasma proteins by IgY microbeads: Meeting the needs of proteomic sample preparation and analysis

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005
Lei Huang
Abstract Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95,99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein,protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics. [source]

Synovial fluid proteins differentiate between the subtypes of juvenile idiopathic arthritis

ARTHRITIS & RHEUMATISM, Issue 6 2010
Margalit E. Rosenkranz
Objective Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA. Methods Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization,time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA). Results The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA. Conclusion Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA. [source]

Isolation of chicken immunoglobulins (IgY) from egg yolk,

BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 3 2003
J. P. Dean Goldring
Abstract Separating individual proteins from complex mixtures of molecules is the basis of many biochemical investigations. The method describes the separation of immunoglobulin Y (IgY) from chicken eggs using a series of physical and chemical separation techniques. The separation is rapid, and the success of each step is readily viewed on an SDS-polyacrylamide gel. IgY identity can be confirmed on a Western blot probed with enzyme-labeled anti-IgY antibodies. The method is a good illustration of protein separation when there is no enzyme activity to follow. [source]

Probing protein colloidal behavior in membrane-based separation processes using spectrofluorometric Rayleigh scattering data

BIOTECHNOLOGY PROGRESS, Issue 3 2010
Rand Elshereef
Abstract One of the primary problems in membrane-based protein separation is membrane fouling. In this study we explored the feasibility of employing Rayleigh light scattering data from fluorescence studies combined with chemometric techniques to determine whether a correlation could be established with membrane fouling phenomena. Membrane flux was measured in a dead-end UF filtration system and the effect of protein solution properties on the flux decline was systematically investigated. A variety of proteins were used as a test case in this study. In parallel, the colloidal behavior of the protein solutions was assessed by employing multiwavelength Rayleigh scattering measurements. To assess the usefulness of Rayleigh scattering measurements for probing the colloidal behavior of proteins, a protein solution of ,-lactoglobulin was used as a base-case scenario. The colloidal behavior of different ,-lactoglobulin solutions was inferred based on published data for this protein, under identical solution conditions, where techniques other than Rayleigh scattering had been used. Using this approach, good agreement was observed between scattering data and the colloidal behavior of this protein. To test the hypothesis that a high degree of aggregation will lead to increased membrane fouling, filtration data was used to find whether the Rayleigh scattering intensity correlated with permeate flux changes. It was found that for protein solutions which were stable and did not aggregate, fouling was reduced and these solutions exhibited reduced Rayleigh scattering. When the aggregation behavior of the solution was favored, significant flux declines occurred and were highly correlated with increased Rayleigh scattering. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]