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Polyacrylamide Gel Electrophoresis (polyacrylamide + gel_electrophoresis)
Kinds of Polyacrylamide Gel Electrophoresis Terms modified by Polyacrylamide Gel Electrophoresis Selected AbstractsComparison of the Electrochemical Behavior of the High Molecular Mass Cadmium Proteins in Arabidopsis thaliana and in Vegetable Plants on Using Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC-PAGE)ELECTROANALYSIS, Issue 1 2006Bernd Kastenholz Abstract In Arabidopsis cytosol (supernatant) and in supernatants of vegetable plants high molecular mass cadmium proteins with molecular mass 200,kDa were isolated by using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE). Because of a different electrochemical behavior of the Cd proteins in Arabidopsis and endive supernatants on using the same PAGE method, it is concluded that the high molecular mass cadmium proteins of Arabidopsis and endive possess different isoelectric points. Consequently, different chemical structures of the Cd proteins with molecular mass 200,kDa are present in Arabidopsis thaliana and in endive. During the electrophoretic separation of vegetable metalloproteins by using the Model 491 Prep Cell from BioRad, electroanalytical processes like electrode reactions may play an important role. [source] Capillary electrophoresis of polycationic poly(amidoamine) dendrimersELECTROPHORESIS, Issue 15 2005Xiangyang Shi Abstract Generation,2 to generation,5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications. [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] Y-position cysteine substitution in type I collagen (,1(I) R888C/p.R1066C) is associated with osteogenesis imperfecta/Ehlers-Danlos syndrome phenotype,,HUMAN MUTATION, Issue 4 2007Wayne A. Cabral Abstract The most common mutations in type I collagen causing types II,IV osteogenesis imperfecta (OI) result in substitution for glycine in a Gly-Xaa-Yaa triplet by another amino acid. We delineated a Y-position substitution in a small pedigree with a combined OI/Ehlers-Danlos Syndrome (EDS) phenotype, characterized by moderately decreased DEXA z-score (,1.3 to ,2.6), long bone fractures, and large-joint hyperextensibility. Affected individuals have an ,1(I)R888C (p.R1066C) substitution in one COL1A1 allele. Polyacrylamide gel electrophoresis (PAGE) of [3H]-proline labeled steady-state collagen reveals slight overmodification of the ,1(I) monomer band, much less than expected for a substitution of a neighboring glycine residue, and a faint ,1(I) dimer. Dimers form in about 10% of proband type I collagen. Dimer formation is inefficient compared to a possible 25%, probably because the SH-side chains have less proximity in this Y-position than when substituting for a glycine. Theoretical stability calculations, differential scanning calorimetry (DSC) thermograms, and thermal denaturation curves showed only weak local destabilization from the Y-position substitution in one or two chains of a collagen helix, but greater destabilization is seen in collagen containing dimers. Y-position collagen dimers cause kinking of the helix, resulting in a register shift that is propagated the full length of the helix and causes resistance to procollagen processing by N-proteinase. Collagen containing the Y-position substitution is incorporated into matrix deposited in culture, including immaturely and maturely cross-linked fractions. In vivo, proband dermal fibrils have decreased density and increased diameter compared to controls, with occasional aggregate formation. This report on Y-position substitutions in type I collagen extends the range of phenotypes caused by nonglycine substitutions and shows that, similar to X- and Y-position substitutions in types II and III collagen, the phenotypes resulting from nonglycine substitutions in type I collagen are distinct from those caused by glycine substitutions. Hum Mutat 28(4), 396,405, 2007. Published 2007 Wiley-Liss, Inc. [source] Biochemical study of resistance to imidacloprid in B biotype Bemisia tabaci from GuatemalaPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 3 2003Frank J Byrne Abstract Systemic uptake bioassays using excised cotton leaves confirmed resistance to imidacloprid in a Guatemalan population of the tobacco whitefly Bemisia tabaci Gennadius. Polyacrylamide gel electrophoresis of naphthyl esterases identified the insects as B-types. Upon collection from the field, resistance was determined to be 58-fold relative to a susceptible strain originating in the Imperial Valley of California. Resistance levels increased to 126-fold in this population during its continuous exposure to systemically treated cotton. In biochemical investigations, there was no detectable NADPH-dependent mixed function oxidase metabolism of 14C-imidacloprid at any time during the selection process. In contrast, microsomal preparations from housefly abdomens readily produced significant amounts of the mono-hydroxy and olefin derivatives of the parent compound. Detoxification of imidacloprid by housefly MFOs may account for reports of lower toxicity of the insecticide towards this insect compared with whiteflies, despite similar binding properties between imidacloprid and the nicotinic acetylcholine receptors in both species. © 2003 Society of Chemical Industry [source] Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleachingPHYSIOLOGIA PLANTARUM, Issue 1 2010Andrea Szenzenstein The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 µmol m,2 s,1photon flux density (PFD) white light and subsequently incubated in total darkness for 4,24 h at 24°C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form,dominating in epicotyls of dark-grown seedling,required 18,24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4,7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units. [source] Control of flatfish sperm motility by CO2 and carbonic anhydraseCYTOSKELETON, Issue 3 2003Kazuo Inaba Abstract Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25,100 mM HCO3,, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3,. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3, -arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/ HCO3, -dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm. Cell Motil. Cytoskeleton 55:174,187, 2003. © 2003 Wiley-Liss, Inc. [source] Effect of celecoxib on cyclooxygenase-2 expression and possible variants in a patient with Barrett's esophagusDISEASES OF THE ESOPHAGUS, Issue 3 2007G. A. Jacobson SUMMARY., Cyclooxygenase-2 (COX-2) expression is increased in metaplastic and dysplastic Barrett's esophageal epithelium and it is thought that selective COX-2 inhibitors could offer hope as chemoprevention therapy. The aim of the study was to investigate the in vivo effect of celecoxib on COX-2 expression in patients with Barrett's esophagus and no recent history of non-steroidal anti-inflammatory drug use. Endoscopic mucosal biopsy specimens were collected at baseline and after 28 days of therapy in a patient treated with celecoxib 200 mg twice daily. Samples were analyzed for COX-2 expression by immunoblot analysis with chemiluminescence detection. COX-2 expression was found to decline 20% and 44% at two different biopsy sites compared to the baseline sample. Longer exposures revealed a number of previously unidentified proteins above and below the 67 kDa COX-2 protein including 38 kDa and 45 kDa proteins which were present only at study completion consistent with up-regulation after celecoxib therapy. Further investigations of the 38 kDa and 45 kDa proteins were undertaken using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with immunoblot and MALDI-TOF (matrix assisted laser desorption ionization , time of flight) analysis but no matches were found and results were inconclusive. Unmatched masses from MALDI-TOF peptide mass fingerprinting were compared with human COX-2 (67 kDa) and COX-2b (39 kDa) using unspecific cleavage. Peptide sequence homology with COX-2 and COX-2b was found for a length of 19 amino acids. Based on immunodetection, molecular weight and equivical MALDI-TOF results, one of these up-regulated proteins may be COX-2b. [source] Comparison of the Electrochemical Behavior of the High Molecular Mass Cadmium Proteins in Arabidopsis thaliana and in Vegetable Plants on Using Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC-PAGE)ELECTROANALYSIS, Issue 1 2006Bernd Kastenholz Abstract In Arabidopsis cytosol (supernatant) and in supernatants of vegetable plants high molecular mass cadmium proteins with molecular mass 200,kDa were isolated by using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE). Because of a different electrochemical behavior of the Cd proteins in Arabidopsis and endive supernatants on using the same PAGE method, it is concluded that the high molecular mass cadmium proteins of Arabidopsis and endive possess different isoelectric points. Consequently, different chemical structures of the Cd proteins with molecular mass 200,kDa are present in Arabidopsis thaliana and in endive. During the electrophoretic separation of vegetable metalloproteins by using the Model 491 Prep Cell from BioRad, electroanalytical processes like electrode reactions may play an important role. [source] Cover Picture: Electrophoresis 10'2010ELECTROPHORESIS, Issue 10 2010Article first published online: 18 MAY 2010 Issue no. 10 is a regular issue comprising 19 manuscripts distributed over four distinct parts. Part I is on microfluidics and miniaturized systems and has 5 articles; Part II is on nucleic acids with 4 articles on restriction endonuclease fingerprinting, mutation detection and DNA separation and detection; Part III has 7 articles on monolithic stationary phases for CEC, single walled carbon nanohorns as pseudo-stationary phases for CEC and EKC, MEEKC, cyclodextrin-modified gold nanoparticles for enantioseparations by CEC, use of divalent dipeptides as counter ions in CE and capillary coating for CE of proteins; and Part IV has 3 articles on proteomics methodologies. Featured articles include: Microfluidic preparative free-flow isoelectric focusing in a triangular channel: System development and characterization ((10.1002/elps.200900577)) Separation and recovery of nucleic acids with improved biological activity by acid-degradable polyacrylamide gel electrophoresis ((10.1002/elps.200900783)) Evaluation of the performance of single-walled carbon nanohorns in capillary electrophoresis ((10.1002/elps.200900628)) The inter- and intra-operator variability in manual spot segmentation and its effect on spot quantitation in two dimensional electrophoresis analysis. ((10.1002/elps.200900674)) [source] Cover Picture: Electrophoresis 14'09ELECTROPHORESIS, Issue 14 2009Article first published online: 28 JUL 200 Issue no. 14 is an Emphasis Issue with 9 articles on various aspects of "Proteins and Proteomics" while the remaining 14 articles are arranged into 4 different parts including "Microfluidics and Miniaturization", "Genotyping and Transcriptomics", "Enantioseparations", and "Nanoparticles and Abused Drugs Analyses". Selected articles are: Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresis ((10.1002/elps.200900026)) 2-D difference in gel electrophoresis combined with Pro-Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets ((10.1002/elps.200800780)) Microvalves actuated sandwich immunoassay on an integrated microfluidic system ((10.1002/elps.200800818)) Chemical gradient-mediated melting curve analysis for genotyping of SNPs ((10.1002/elps.200800729)) [source] Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresisELECTROPHORESIS, Issue 14 2009Jingdan Liang Abstract Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps. [source] Cover Picture: Electrophoresis 4/2008ELECTROPHORESIS, Issue 4 2008Article first published online: 22 FEB 200 This special issue on capillary electrochromatography (CEC) and electrokinetic capillary chromatography (EKC) provides the reader with the latest developments and improvements in these two closely related micro-column separation techniques. Issue 4 also offers one Fast Track article describing particularly important investigations in electrophoresis: Identification of unknown protein complex members by radiolocalization and analysis of low-abundance complexes resolved using native polyacrylamide gel electrophoresis Mahuya Bose, Brian P. Adams, Randy M. Whittal, Himangshu S. Bose [source] Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresisELECTROPHORESIS, Issue 7 2006Zora Nováková Abstract The nucleus is a highly structured organelle with distinct compartmentalization of specific functions. To understand the functions of these nuclear compartments, detailed description of protein complexes which form these structures is of crucial importance. We explored therefore the potential of blue native PAGE (BN-PAGE) method for the separation of nuclear protein complexes. We focused on (i),solubility and stability of nuclear complexes under conditions prerequisite for the separation by BN-PAGE, (ii),improved separation of native nuclear protein complexes using 2-D colorless native/blue native PAGE (CN-/BN-PAGE), and (iii),mass spectrometric analysis of protein complexes which were isolated directly from native 1-D or from 2-D CN/BN-PAGE gels. The suitability of BN-PAGE for nuclear proteomic research is demonstrated by the successful separation of polymerase,I and polymerase,II complexes, and by mass spectrometric determination of U1 small nuclear ribonucleoprotein particle composition. Moreover, practical advice for sample preparation is provided. [source] A new multiphasic buffer system for benzyldimethyl- n -hexadecylammonium chloride polyacrylamide gel electrophoresis of proteins providing efficient stackingELECTROPHORESIS, Issue 2 2006Michael L. Kramer Dr. Abstract Acidic PAGE systems using cationic detergents such as benzyldimethyl- n -hexadecylammonium chloride (16-BAC) or CTAB have proven useful for the detection of methoxy esters sensitive to alkaline pH, resolving basic proteins such as histones and membrane proteins. However, the interesting phosphate-based system suffered from poor stacking, resulting in broadened bands and long running times. Therefore, a new 16-BAC PAGE system based on the theory of moving boundary electrophoresis with properties comparable to the classical SDS-PAGE system was designed. As a result a new multiphasic analytical 16-BAC PAGE system providing efficient stacking and significantly shorter running times is presented here. It is based on acetic acid and methoxyacetic acid as common ion constituents. This PAGE system takes advantage of the additional counterstacking effect due to a cross boundary electrophoresis system resulting from the selected buffer constituents. Furthermore, the concentration of 16-BAC was optimized by determining its previously unknown CMC. Due to efficient focusing of the introduced tracking dye, methyl green, termination of electrophoresis can now be more easily followed as compared to the Schlieren line. [source] Separation of proteins with a molecular mass difference of 2,kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: Application to the isolation of 24,kDa human growth hormoneELECTROPHORESIS, Issue 23 2005Juan J. Bustamante Abstract A method for separating proteins with a molecular mass difference of 2,kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24,kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2,kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24,kDa hGH. The 22 and 24,kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13,18% linear gradient gel, and a 15,10% linear inverted gradient gel. Fractions containing purified 24,kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2,kDa. [source] Analysis of poly(amidoamine)-succinamic acid dendrimers by slab-gel electrophoresis and capillary zone electrophoresisELECTROPHORESIS, Issue 15 2005Xiangyang Shi Abstract Ethylenediamine (EDA)-core poly(amidoamine) (PAMAM) succinamic acid dendrimers (Ex.SAH, where x refers to the generation) were synthesized and analyzed by polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), potentiometric acid-base titration, and capillary zone electrophoresis (CZE). Various generations (E1.SAH,E7.SAH) PAMAMs and a succinamic acid terminated core-shell tecto(dendrimer) (E5(E3.SAH)n) were first analyzed by PAGE. PAGE results show that the relative mobilities of generation,2 to generation,7 dendrimers decreased with the increasing number of generations. The molecular mass of a generation,5 core generation,3 shell tecto(dendrimer) (denoted as E5(E3.SAH)n) was determined to be between the Mw of E6.SAH and E7.SAH. CZE analysis allowed the evaluation of electrophoretic properties of given-generation dendrimers. The electrophoretic mobilities of individual generations PAMAM polyanions are similar, indicating that the separation mainly depends on their approximately identical charge/mass ratio. The E5(E3.SAH)n tectodendrimer had a lower electrophoretic mobility, which was consistent with its lower charge/mass ratio. The combination of PAGE and CZE analysis provides an alternative and effective way to characterize this group of PAMAM-succinamic acid dendrimers. [source] Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separationsELECTROPHORESIS, Issue 7-8 2004Yi Zhu Abstract Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains. [source] Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresisELECTROPHORESIS, Issue 16 2003Gianfranco Mamone Abstract We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five ,-, fifteen ,s1 -, ten ,s2 -, and four ,-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to ,-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single ,-CN component. The phosphate group on site Ser12 of tryptic peptide 8,22 of most phosphorylated ,s1 -CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two ,-CN components was determined by means of MS/MS analysis. [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] Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: Comparative proteomic studies on rat retina and mouse brainELECTROPHORESIS, Issue 4 2003Simone König Abstract A long-chain derivative of 1,3-dioxolane sodium propyloxy sulfate, with similar denaturing and electrophoretic properties as SDS, and facilitated protein identification following polyacrylamide gel electrophoresis (PAGE) for Coomassie-stained protein bands, has been tested. Comparative acid-labile surfactant/sodium dodecyl sulfate two-dimensional (ALS/SDS 2-D)-PAGE experiments of lower abundant proteins from the proteomes of regenerating rat retina and mouse brain show that peptide recovery for mass spectrometry (MS) mapping is significantly enhanced using ALS leading to more successful database searches. ALS may influence some procedures in proteomic analysis such as the determination of protein content and methods need to be adjusted to that effect. The promising results of the use of ALS in bioanalytics call for detailed physicochemical investigations of surfactant properties. [source] Background-free, fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes, zincon and ethyl violetELECTROPHORESIS, Issue 24 2002Jung-Kap Choi Abstract A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1,1.5 h. The detection limit of this method is 8,15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes. [source] Two-dimensional gel analysis of stress proteins identified in Chironomus flaviplumus (Diptera: Chironomidae) exposed to 4-nonylphenolENTOMOLOGICAL RESEARCH, Issue 3 2010Myoung Chul KIM Abstract Toxicity of 4-nonylphenol (4-NP) in the Chironomidae Chironomus flaviplumus was analyzed using a proteomics approach that involved identifying proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Proteome analysis of 4-NP-treated samples on silver stained gels found alterations in the expression levels of three proteins compared with control samples. Hsp70 proteins, so-called stress proteins, were studied in Chironomus flaviplumus exposed to different concentrations of 4-nonylphenol (0, 30, 100, 150, 300 and 600 mg/L) in the laboratory and in the field in captured animals from site 1 (1 km from a chemical factory) and site 3 (16 km from a chemical factory). Hsp70 proteins were found in all samples tested, including controls, but differed in their expression levels. At more polluted sites (site 1), the samples treated with higher concentrations of 4-NP more strongly expressed Hsp70. 2-D spots were induced or enhanced in gels following injection of 4-NP. Therefore, the induction of stress protein expression in Chironomus flaviplumus, in particular Hsp70, can be used as a biomarker for the evaluation of environmental conditions. [source] Important region in the ,-spectrin C -terminus for spectrin tetramer formationEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2002Bing-Hao Luo Abstract: Many hereditary hemolytic anemias are due to spectrin mutations at the C -terminal region of ,-spectrin (the ,C region) that destabilize spectrin tetramer formation. However, little is known about the ,C region of spectrin. We have prepared four recombinant ,-peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C -terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N -terminal region ,-peptide (Sp,1,156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the ,-peptide. Circular dichroism results showed that the unassociated helices in both ,- and ,-peptides became associated, presumably to form a helical bundle, for those ,-peptides that formed an ,, complex, but not for those ,-peptides that did not form an ,, complex. In addition, upon association, an increase in the ,-helical content was observed. These results showed that the ,-peptides ending prior to residue 2072 (Thr) would not associate with ,-peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C -terminal segment of ,-spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C -terminal region ending with residue 2072 is important for its association with ,-spectrin in forming tetramers. [source] An assay system for the detection of phospholipase C activityEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 10 2003Markus Durban Abstract Phospholipase C (PLC, EC 3.1.4.3) enzymes specifically hydrolyze the C-O-P-bond in phospholipids, yielding sn -1, 2(2, 3)-diglycerides and a phosphate residue bearing the corresponding head group. Biochemical characterization of PLC requires methods for determination of activity. During characterization and purification, proteins are separated by polyacrylamide gel electrophoresis (PAGE). For direct identification and visualization of PLC, a new assay for activity staining in native and renatured SDS-PAGE is described. Incubation of a gel containing an active PLC in the presence of ,-naphthylphosphorylcholine leads to ,-naphthol formation. This reacts with the diazonium salt Fast Red, forming a red dye which allows clear determination of PLC purity, molecular weight and substrate specificity. The assay was verified using commercially available PC-PLC and new PC-PLC-producing Bacillus cereus strains. The substrate ,-NPC was prepared by chemical synthesis at an overall yield of 12%. [source] Distribution of SIBLING proteins in the organic and inorganic phases of rat dentin and boneEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2 2008Bingzhen Huang The SIBLING protein family is a group of non-collagenous proteins (NCPs) that includes dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN). In the present study, we compared these four proteins in different phases of rat dentin and bone. First, we extracted NCPs in the unmineralized matrices and cellular compartments using guanidium-HCl (G1). Second, we extracted NCPs closely associated with hydroxyapatite using an EDTA solution (E). Last, we extracted the remaining NCPs again with guanidium-HCl (G2). Each fraction of Q-Sepharose ion-exchange chromatography was analyzed using sodium dodecyl sulfate,polyacrylamide gel electrophoresis (SDS,PAGE), Stains-All stain, and with western immunoblotting. In dentin, the NH2 -terminal fragment of DSPP and its proteoglycan form were primarily present in the G1 extract, whereas the COOH-terminal fragment of DSPP was present exclusively in the E extract. The processed NH2 -terminal fragment of DMP1 was present in G1 and E extracts, whereas the COOH-terminal fragment of DMP1 existed mainly in the E extract. Bone sialoprotein was present in all three extracts of dentin and bone, whereas OPN was present only in the G1 and E extracts of bone. The difference in the distribution of the SIBLING proteins between organic and inorganic phases supports the belief that these molecular species play different roles in dentinogenesis and osteogenesis. [source] A method for the analysis of milk and egg allergens for the atopy patch testEXPERIMENTAL DERMATOLOGY, Issue 10 2009Cinzia Ballabio Abstract:, The patch test with food antigens (atopy patch test, APT) has been reported as a more specific method than prick or RAST for the early detection of cow's milk and/or egg sensitizations in children. Standardization of APT extracts is a major issue on the road towards full clinical exploitation of this assay. Here, we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to characterize sensitivity and specificity of commercial preparations of APT for milk and egg allergies, which are expected to improve the reliability of this test, when compared with fresh food allergen sources. We found that: (i) SDS-PAGE is an appropriate technique for quality control of APT and (ii) commercial milk and egg APT are equivalent to fresh food preparations in terms of allergen content. Clinical trials aimed at characterizing sensitivity and specificity of APT in the diagnosis of food allergy in children will benefit from this technique. [source] Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp.FEBS JOURNAL, Issue 7 2004SIB1 in cold-adaptation A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 °C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 °C compared to that at 20 °C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N -succinyl-Ala-Leu-Pro-Phe- p -nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 °C with a kcat/Km value of 0.87 µm,1·s,1. When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T1 refolding assay at 10 and 20 °C, the protein exhibited higher activity at 10 °C with a kcat/Km value of 0.50 µm,1·s,1. These kcat/Km values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria. [source] Two W-containing formate dehydrogenases (CO2 -reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidansFEBS JOURNAL, Issue 11 2003Frank A. M. De Bok Two formate dehydrogenases (CO2 -reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2 -reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U·mg,1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U·mg,1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2 -reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U·mg,1 and 89 U·mg,1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 ± 3, 56 ± 2 and 19 ± 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 ± 3 kDa and a 33 ± 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters. [source] The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosisFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2006Henrik Chart Abstract The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis. [source] | |||||||||||||||||||||||||||||||||||||