Native Polyacrylamide Gel Electrophoresis (native + polyacrylamide_gel_electrophoresis)

Distribution by Scientific Domains

Kinds of Native Polyacrylamide Gel Electrophoresis

  • blue native polyacrylamide gel electrophoresis


  • Selected Abstracts


    Important region in the ,-spectrin C -terminus for spectrin tetramer formation

    EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2002
    Bing-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]


    Immunolocalization of 1,3-,-Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

    JOURNAL OF PHYTOPATHOLOGY, Issue 5 2010
    Yongting Yu
    Abstract The distribution of extracellular 1,3-,-glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3-,-glucanase secreted by the pathogenic fungus. A specific antibody of 1,3-,-glucanase, anti-GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3-,-glucanase of Ggt, but not for 1,3-,-glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3-,-glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen-infected wheat roots with anti-1,3-,-glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non-inoculated wheat roots. The results indicate that 1,3-,-glucanase secreted by Ggt may be involved in pathogenesis of the take-all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers. [source]


    Insecticide resistance spectra and resistance mechanisms in populations of Japanese encephalitis vector mosquitoes, Culex tritaeniorhynchus and Cx. gelidus, in Sri Lanka

    MEDICAL AND VETERINARY ENTOMOLOGY, Issue 4 2000
    S. H. P. P. Karunaratne
    Summary Culex tritaeniorhynchus Giles and Cx. gelidus Theobald (Diptera: Culicidae), both vectors of Japanese encephalitis, were collected in 1984 and 1998 from two disease endemic localities in Sri Lanka: Anaradhapura and Kandy. Using wild-caught adult mosquitoes from light traps, log dosage-probit mortality curves for insecticide bioassays were obtained for three insecticides: malathion (organophosphate), propoxur (carbamate) and permethrin (pyrethroid). LD50 values showed that, in 1998, Cx. tritaeniorhynchus was ,100-fold more resistant to malathion and 10-fold more resistant to propoxur than was Cx. gelidus. This difference was attributed to Cx. tritaeniorhynchus breeding mostly in irrigated rice paddy fields, where it would have been exposed to pesticide selection pressure, whereas Cx. gelidus breeds in other types of aquatic habitats less prone to pesticide applications. Resistance in Cx. tritaeniorhynchus increased between 1984 and 1998, whereas Cx. gelidus remained predominantly susceptible. Propoxur inhibition of acetylcholinesterase (AChE) activity (the target site of organophosphates and carbamates) indicated that in 1998, frequencies of insensitive AChE-based resistance were 9% in Cx. gelidus and 2,23% in Cx. tritaeniorhynchus, whereas in 1984 this resistance mechanism was detected only in 2% of the latter species from Anaradhapura. The AChE inhibition coefficient (ki) with propoxur was 1.86 ± 0.24 × 105 m,1 min,1 for Cx. tritaeniorhynchus from Anaradhapura in 1998. Both species were tested for activity levels of detoxifying glutathione S-trans- ferases (GSTs) and malathion-specific as well as general carboxylesterases. High activities of GSTs and carboxylesterases were detected in Cx. tritaeniorhynchus but not Cx. gelidus. Malathion-specific carboxylesterase was absent from both species. Native polyacrylamide gel electrophoresis resolved two elevated general carboxylesterases, CtrEst,1 and CtrEst,1, from Cx. tritaeniorhynchus and none from Cx. gelidus. CtrEst,1 was the most intensely staining band. Gel inhibition experiments showed that both elevated esterases were inhibited by organophosphates and carbamates but not by pyrethroids. The major elevated esterase CtrEst,1 was partially purified (15-fold) by sequential Q-Sepharose and phenyl Sepharose column chromatography. The bimolecular rate constant (ka) and the deacylation rate constant (k3) for the malaoxon/ enzyme interaction were 9.9 ± 1.1 × 103 m,1 min,1 and 3.5 ± 0.05 × 10,4m,1 min,1, respectively, demonstrating that the role of this enzyme in organophosphorus insecticide resistance is sequestration. [source]


    Ascorbate-dependent hydrogen peroxide detoxification and ascorbate regeneration during germination of a highly productive maize hybrid: Evidence of an improved detoxification mechanism against reactive oxygen species

    PHYSIOLOGIA PLANTARUM, Issue 1 2000
    Laura De Gara
    Ascorbate content and the activities of some key enzymes involved in the detoxification from reactive oxygen species were investigated in germinated embryos of two Zea mays L. inbred lines (B73 and Mo17) and of their heterotic F1 hybrid (B73×Mo17). The F1 hybrid showed a higher ascorbate biosynthetic capability owing to a higher activity of l -galactono- , -lactone dehydrogenase (EC 1.6.5.4), the last enzyme in ascorbate biosynthesis. Ascorbate peroxidase (EC 1.11.1.11), ascorbate free radical reductase (EC 1.6.5.4) and dehydroascorbate reductase (EC 1.8.5.1) activities were much higher in the F1 hybrid than in either inbred line, whereas catalase (EC 1.11.1.6) activity was similar in the three genotypes. Native polyacrylamide gel electrophoresis (PAGE) analysis showed three forms of cytosolic ascorbate peroxidase, both in parental lines and in the F1 hybrid. On the other hand, a complex pattern of proteins with dehydroascorbate reductase activity was observed, with the hybrid combining the different dehydroascorbate-reducing proteins expressed by the inbred lines. The possible involvement of the enzymes of the ascorbate system in the phenomenon of hybrid vigour is discussed. [source]


    Low mutant load of mitochondrial DNA G13513A mutation can cause Leigh's disease

    ANNALS OF NEUROLOGY, Issue 4 2003
    Denise M. Kirby BSc(Hons)
    Respiratory chain complex I deficiency is a common cause of Leigh's disease (LD) and can be caused by mutations in genes encoded by either nuclear or mitochondrial DNA (mtDNA). Most pathogenic mtDNA mutations act recessively and only cause disease when present at high mutant loads (typically >90%) in tissues such as muscle and brain. Two mitochondrial DNA mutations in complex I subunit genes, G14459A in ND6, and T12706C in ND5, have been associated with complex I deficiency and LD. We report another ND5 mutation, G13513A, in three unrelated patients with complex I deficiency and LD. The G13513A mutation was present at mutant loads of approximately 50% or less in all tissues tested, including multiple brain regions. The threshold mutant load for causing a complex I defect in cultured cells was approximately 30%. Blue Native polyacrylamide gel electrophoresis showed that fibroblasts with 45% G13513A mutant load had approximately 50% of the normal amount of fully assembled complex I. Fibroblasts with greater than 97% of the ND6 G14459A mutation had only 20% fully assembled complex I, suggesting that both mutations disrupt complex I assembly or turnover. We conclude that the G13513A mutation causes a complex I defect when present at unusually low mutant load and may act dominantly. [source]


    Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

    ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010
    Marsha M. Wheeler
    Abstract Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when ,-naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub-cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)-derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional-genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc. [source]


    Cover Picture: Electrophoresis 14'09

    ELECTROPHORESIS, Issue 14 2009
    Article 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 electrophoresis

    ELECTROPHORESIS, Issue 14 2009
    Jingdan 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 4/2008

    ELECTROPHORESIS, Issue 4 2008
    Article 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 electrophoresis

    ELECTROPHORESIS, Issue 7 2006
    Zora 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]


    Detection of a homotetrameric structure and protein,protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights

    FEMS YEAST RESEARCH, Issue 1 2010
    Clayton Luiz Borges
    Abstract Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role in fungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was overexpressed in bacteria and a polyclonal antibody to this protein was produced. We identified a 180-kDa protein species reactive to the antibody produced in mice against the P. brasiliensis recombinant purified formamidase of 45 kDa. The 180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide mass fingerprinting using MS. The identical mass spectra generated by the 180 and the 45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formamidase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights into formamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism. [source]


    The interaction between ,S, the stationary phase , factor, and the core enzyme of Escherichia coli RNA polymerase

    GENES TO CELLS, Issue 3 2002
    Frédéric Colland
    Background: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure ,2,,,) associated with one of the , subunits, required for promoter recognition. Different , factors compete for core binding. Among the seven , factors present in E. coli, ,70 controls gene transcription during the exponential phase, whereas ,S regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled ,S and ,70, we compared the affinities of both , factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. Results: Using native polyacrylamide gel electrophoresis, we demonstrate that ,S binds to the core enzyme with fivefold reduced affinity compared to ,70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of ,S, while increasing the accessibility in region 4.1 of ,S. We have also analysed the positioning of ,S on the holoenzyme by the proximity-dependent protein cleavage method using ,S derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the , and ,, subunits, but not ,. Regions 2.5, 3.1 and 3.2 of ,S are close to both , and ,, subunits, in agreement with iron chelate protein footprinting data. Conclusions: A comparison between these results using ,S and previous data from ,70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2. [source]


    Low complex I content explains the low hydrogen peroxide production rate of heart mitochondria from the long-lived pigeon, Columba livia

    AGING CELL, Issue 1 2010
    Adrian J. Lambert
    Summary Across a range of vertebrate species, it is known that there is a negative association between maximum lifespan and mitochondrial hydrogen peroxide production. In this report, we investigate the underlying biochemical basis of the low hydrogen peroxide production rate of heart mitochondria from a long-lived species (pigeon) compared with a short-lived species with similar body mass (rat). The difference in hydrogen peroxide efflux rate was not explained by differences in either superoxide dismutase activity or hydrogen peroxide removal capacity. During succinate oxidation, the difference in hydrogen peroxide production rate between the species was localized to the ,pH-sensitive superoxide producing site within complex I. Mitochondrial ,pH was significantly lower in pigeon mitochondria compared with rat, but this difference in ,pH was not great enough to explain the lower hydrogen peroxide production rate. As judged by mitochondrial flavin mononucleotide content and blue native polyacrylamide gel electrophoresis, pigeon mitochondria contained less complex I than rat mitochondria. Recalculation revealed that the rates of hydrogen peroxide production per molecule of complex I were the same in rat and pigeon. We conclude that mitochondria from the long-lived pigeon display low rates of hydrogen peroxide production because they have low levels of complex I. [source]


    Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasing

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003
    J. Shaun Lott
    The protein PAE2307 is a member of a protein family of unknown function which is conserved among a number of bacterial and archaeal species. The protein was overexpressed in Escherichia coli, purified and crystallized in two crystal forms. The prevalent form was twinned, but the other diffracted to 1.45,Ĺ resolution. The non-twinned crystals proved difficult to reproduce, so screening of potential heavy-atom derivatives by native polyacrylamide gel electrophoresis was used to establish suitable derivatization conditions. This process enabled the production of a K2Pt(NO2)4 derivative that was used to collect a single-wavelength anomalous diffraction (SAD) data set from the only available crystal. Phase information of high quality was obtained, enabling the calculation of an interpretable electron-density map. [source]


    Biochemical mechanisms of insecticide resistance in the diamondback moth (DBM), Plutella xylostella L. (Lepidopterata: Yponomeutidae), in the Sydney region, Australia

    AUSTRALIAN JOURNAL OF ENTOMOLOGY, Issue 4 2009
    Vincent Y Eziah
    Abstract Following the detection of resistant diamondback moth (DBM) populations to synthetic pyrethroid, organophosphorus and indoxacarb insecticides in the Sydney Basin, a study of the major biochemical mechanisms was conducted to determine the type of resistance in these populations. The activity of cytochrome P450 monooxygenases increased two- to sixfold when compared with the susceptible strain. Up to a 1.9-fold increase in esterase activity in resistant strains compared with the susceptible strain was observed. In vitro inhibition studies showed that profenofos, methamidophos and chlorpyrifos strongly inhibited the esterases while permethrin and esfenvalerate resulted in less than 30% inhibition. Qualitative analysis of the esterases using native polyacrylamide gel electrophoresis showed four bands in both the susceptible and resistant individuals with more intense staining in the resistant individuals. The development of these bands was inhibited by methamidophos and chlorpyrifos pretreatment of the protein extract while permethrin and esfenvalerate did not exhibit this effect. Glutathione S-transferase (GST) activity was significantly higher in two field populations compared with the remaining populations. Overall, the study showed that the mechanisms of insecticide resistance in the DBM populations in the area studied were due to cytochrome P450 monooxygenases, esterase and GSTs, and possibly other non-metabolic mechanisms that were not investigated in the present study. [source]