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[PMID]:29360877
[Au] Autor:Verma V; Kaur C; Grover P; Gupta A; Chaudhary VK
[Ad] Endereço:Centre for Innovation in Infectious Disease Research, Education and Training (CIIDRET), University of Delhi South Campus, New Delhi, India.
[Ti] Título:Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.
[So] Source:PLoS One;13(1):e0191315, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.
[Mh] Termos MeSH primário: Proteínas de Bactérias/isolamento & purificação
Proteínas de Bactérias/metabolismo
Biotina/metabolismo
Ensaio de Imunoadsorção Enzimática/métodos
Biblioteca de Peptídeos
Testes Sorológicos/métodos
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Proteínas de Bactérias/química
Proteínas de Bactérias/genética
Biotinilação
Carbono-Nitrogênio Ligases/metabolismo
Clonagem Molecular
Citosol/metabolismo
Escherichia coli/citologia
Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Vetores Genéticos/genética
Indicadores e Reagentes/metabolismo
Mycobacterium tuberculosis/genética
Proteínas Repressoras/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Escherichia coli Proteins); 0 (Indicators and Reagents); 0 (Peptide Library); 0 (Repressor Proteins); 6SO6U10H04 (Biotin); EC 6.3.- (Carbon-Nitrogen Ligases); EC 6.3.4.15 (birA protein, E coli)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180124
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0191315


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[PMID]:28470616
[Au] Autor:Gräslund S; Savitsky P; Müller-Knapp S
[Ad] Endereço:Structural Genomics Consortium, Department of Biochemistry and Biophysics, Karolinska Institutet, Tomtebodavägen 23a, Gamma:6, 171 65, Solna, Sweden. susanne.graslund@ki.se.
[Ti] Título:In Vivo Biotinylation of Antigens in E. coli.
[So] Source:Methods Mol Biol;1586:337-344, 2017.
[Is] ISSN:1940-6029
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Site-specific biotinylation of proteins is often the method of choice to enable efficient immobilization of a protein on a surface without interfering with protein folding. The tight interaction of biotin and streptavidin is frequently used to immobilize an antigen during phage display selections of binders. Here we describe a method of in vivo biotinylation of proteins during expression in E. coli, by tagging the protein with the short biotin acceptor peptide sequence, Avi tag, and co-expression of the E. coli biotin ligase (BirA) resulting in precise biotinylation of a specific lysine residue in the tag.
[Mh] Termos MeSH primário: Antígenos/química
Antígenos/genética
Escherichia coli/genética
Proteínas Imobilizadas/química
Proteínas Imobilizadas/genética
[Mh] Termos MeSH secundário: Animais
Biotina/química
Biotinilação
Carbono-Nitrogênio Ligases/química
Carbono-Nitrogênio Ligases/genética
Clonagem Molecular/métodos
Escherichia coli/química
Proteínas de Escherichia coli/química
Proteínas de Escherichia coli/genética
Expressão Gênica
Vetores Genéticos/genética
Seres Humanos
Proteínas Repressoras/química
Proteínas Repressoras/genética
Estreptavidina/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antigens); 0 (Escherichia coli Proteins); 0 (Immobilized Proteins); 0 (Repressor Proteins); 6SO6U10H04 (Biotin); 9013-20-1 (Streptavidin); EC 6.3.- (Carbon-Nitrogen Ligases); EC 6.3.4.15 (birA protein, E coli)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180220
[Lr] Data última revisão:
180220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170505
[St] Status:MEDLINE
[do] DOI:10.1007/978-1-4939-6887-9_22


  3 / 4461 MEDLINE  
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[PMID]:28461655
[Au] Autor:Mata J; Wise JA
[Ad] Endereço:Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom jm593@cam.ac.uk.
[Ti] Título:4-Thiouridine Labeling to Analyze mRNA Turnover in .
[So] Source:Cold Spring Harb Protoc;2017(5):pdb.prot091645, 2017 May 01.
[Is] ISSN:1559-6095
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Traditionally, the half-lives of mRNAs were measured after inhibition of transcription to allow decay of the preexisting population. The protocol presented here is a more recently developed strategy in which mRNA turnover is analyzed by measuring the decline in levels of newly synthesized RNA labeled with 4-thiouridine (4sU) during a brief pulse. After RNA extraction, the 4sU is biotinylated and the labeled species are purified using streptavidin beads. DNA microarrays can then be used to compare this population with total RNA, allowing half-lives to be calculated.
[Mh] Termos MeSH primário: Marcadores de Afinidade
RNA Fúngico/metabolismo
RNA Mensageiro/metabolismo
Schizosaccharomyces/metabolismo
Tiouridina
[Mh] Termos MeSH secundário: Antimetabólitos
Biotinilação
Meia-Vida
Indicadores e Reagentes
Análise de Sequência com Séries de Oligonucleotídeos
RNA Fúngico/biossíntese
RNA Mensageiro/análise
RNA Mensageiro/biossíntese
Estreptavidina
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Affinity Labels); 0 (Antimetabolites); 0 (Indicators and Reagents); 0 (RNA, Fungal); 0 (RNA, Messenger); 13957-31-8 (Thiouridine); 9013-20-1 (Streptavidin)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180129
[Lr] Data última revisão:
180129
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170503
[St] Status:MEDLINE
[do] DOI:10.1101/pdb.prot091645


  4 / 4461 MEDLINE  
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[PMID]:29236386
[Au] Autor:Galkin OY; Besarab AB; Lutsenko TN
[Ti] Título:Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60)
[So] Source:Ukr Biochem J;89(1):22-30, 2017 Jan-Feb.
[Is] ISSN:2409-4943
[Cp] País de publicação:Ukraine
[La] Idioma:eng
[Ab] Resumo:The goal of this work was to study sensitivity and specificity of the developed ELISA set for the identification of IgG antibodies against Chlamydia trachomatis HSP-60 (using biotinylated tyramine-based signal amplification system). The study was conducted using a panel of characterized sera, as well as two reference ELISA sets of similar purpose. According to the results of ELISA informative value parameters, the ELISA we have developed showed the highest specificity and sensitivity parameters (no false negative or false positive results were registered). In 4 out of 15 intralaboratory panel serum samples initially identified as negative, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive. The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples. Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result. High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven. Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant ELISA-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents.
[Mh] Termos MeSH primário: Anticorpos Antibacterianos/análise
Antígenos de Bactérias/sangue
Chaperonina 60/sangue
Infecções por Chlamydia/diagnóstico
Chlamydia trachomatis/imunologia
Ensaio de Imunoadsorção Enzimática/métodos
Imunoglobulina G/análise
[Mh] Termos MeSH secundário: Anticorpos Anti-Idiotípicos/química
Anticorpos Antibacterianos/sangue
Anticorpos Antibacterianos/imunologia
Complexo Antígeno-Anticorpo/química
Antígenos de Bactérias/imunologia
Biotinilação
Chaperonina 60/imunologia
Infecções por Chlamydia/sangue
Infecções por Chlamydia/imunologia
Infecções por Chlamydia/microbiologia
Chlamydia trachomatis/química
Ensaio de Imunoadsorção Enzimática/normas
Reações Falso-Negativas
Seres Humanos
Soros Imunes/química
Imunoglobulina G/sangue
Imunoglobulina G/imunologia
Sensibilidade e Especificidade
Tiramina/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antibodies, Anti-Idiotypic); 0 (Antibodies, Bacterial); 0 (Antigen-Antibody Complex); 0 (Antigens, Bacterial); 0 (Chaperonin 60); 0 (Immune Sera); 0 (Immunoglobulin G); X8ZC7V0OX3 (Tyramine)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180116
[Lr] Data última revisão:
180116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171214
[St] Status:MEDLINE
[do] DOI:10.15407/ubj89.01.022


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[PMID]:29235323
[Au] Autor:Grinenko TV; Kapustianenko LG; Yatsenko TA; Yusova OI; Rybachuk VN
[Ti] Título:Plasminogen fragments K 1-3 and K 5 bind to different sites in fibrin fragment DD.
[So] Source:Ukr Biochem J;88(3):36-45, 2016 May-Jun.
[Is] ISSN:2409-4943
[Cp] País de publicação:Ukraine
[La] Idioma:eng
[Ab] Resumo:Specific plasminogen-binding sites of fibrin molecule are located in Аα148-160 regions of C-terminal domains. Plasminogen interaction with these sites initiates the activation process of proenzyme and subsequent fibrin lysis. In this study we investigated the binding of plasminogen fragments K 1-3 and K 5 with fibrin fragment DD and their effect on Glu-plasminogen interaction with DD. It was shown that the level of Glu-plasminogen binding to fibrin fragment DD is decreased by 50-60% in the presence of K 1-3 and K 5. Fragments K 1-3 and K 5 have high affinity to fibrin fragment DD (Kd is 0.02 for K 1-3 and 0.054 µÐœ for K 5). K 5 interaction is independent and K 1-3 is partly dependent on C-terminal lysine residues. K 1-3 interacts with complex of fragment DD-immobilized K 5 as well as K 5 with complex of fragment DD-immobilized K 1-3. The plasminogen fragments do not displace each other from binding sites located in fibrin fragment DD, but can compete for the interaction. The results indicate that fibrin fragment DD contains different binding sites for plasminogen kringle fragments K 1-3 and K 5, which can be located close to each other. The role of amino acid residues of fibrin molecule Аα148-160 region in interaction with fragments K 1-3 and K 5 is discussed.
[Mh] Termos MeSH primário: Produtos de Degradação da Fibrina e do Fibrinogênio/química
Fibrinogênio/química
Fragmentos de Peptídeos/química
Plasminogênio/química
[Mh] Termos MeSH secundário: Sítios de Ligação
Ligação Competitiva
Biotinilação
Seres Humanos
Cinética
Ligação Proteica
Domínios e Motivos de Interação entre Proteínas
Proteólise
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Fibrin Fibrinogen Degradation Products); 0 (Peptide Fragments); 0 (fibrin fragment D); 9001-32-5 (Fibrinogen); 9001-91-6 (Plasminogen)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180116
[Lr] Data última revisão:
180116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171214
[St] Status:MEDLINE
[do] DOI:10.15407/ubj88.03.036


  6 / 4461 MEDLINE  
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[PMID]:28455373
[Au] Autor:Matsuda K; Mikami T; Oki S; Iida H; Andrabi M; Boss JM; Yamaguchi K; Shigenobu S; Kondoh H
[Ad] Endereço:Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan.
[Ti] Título:ChIP-seq analysis of genomic binding regions of five major transcription factors highlights a central role for ZIC2 in the mouse epiblast stem cell gene regulatory network.
[So] Source:Development;144(11):1948-1958, 2017 06 01.
[Is] ISSN:1477-9129
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:To obtain insight into the transcription factor (TF)-dependent regulation of epiblast stem cells (EpiSCs), we performed ChIP-seq analysis of the genomic binding regions of five major TFs. Analysis of biotinylated ZIC2, OTX2, SOX2, POU5F1 and POU3F1 binding in EpiSCs identified several new features. (1) Megabase-scale genomic domains rich in ZIC2 peaks and genes alternate with those rich in POU3F1 but sparse in genes, reflecting the clustering of regulatory regions that act at short and long-range, which involve binding of ZIC2 and POU3F1, respectively. (2) The enhancers bound by ZIC2 and OTX2 prominently regulate TF genes in EpiSCs. (3) The binding sites for SOX2 and POU5F1 in mouse embryonic stem cells (ESCs) and EpiSCs are divergent, reflecting the shift in the major acting TFs from SOX2/POU5F1 in ESCs to OTX2/ZIC2 in EpiSCs. (4) This shift in the major acting TFs appears to be primed by binding of ZIC2 in ESCs at relevant genomic positions that later function as enhancers following the disengagement of SOX2/POU5F1 from major regulatory functions and subsequent binding by OTX2. These new insights into EpiSC gene regulatory networks gained from this study are highly relevant to early stage embryogenesis.
[Mh] Termos MeSH primário: Imunoprecipitação da Cromatina
Regulação da Expressão Gênica no Desenvolvimento
Redes Reguladoras de Genes
Camadas Germinativas/citologia
Células-Tronco Embrionárias Murinas/metabolismo
Análise de Sequência de RNA
Fatores de Transcrição/metabolismo
[Mh] Termos MeSH secundário: Animais
Sítios de Ligação/genética
Biotinilação
Genoma
Camadas Germinativas/metabolismo
Seres Humanos
Camundongos
Células-Tronco Embrionárias Murinas/citologia
Fator 3 de Transcrição de Octâmero/metabolismo
Fatores de Transcrição Otx/metabolismo
Ligação Proteica
Fatores de Transcrição SOXB1/metabolismo
Fatores de Transcrição/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Octamer Transcription Factor-3); 0 (Otx Transcription Factors); 0 (Otx2 protein, mouse); 0 (Pou5f1 protein, mouse); 0 (SOXB1 Transcription Factors); 0 (Transcription Factors); 0 (Zic2 protein, mouse)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:180112
[Lr] Data última revisão:
180112
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170430
[St] Status:MEDLINE
[do] DOI:10.1242/dev.143479


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[PMID]:29253870
[Au] Autor:Sasidhar MV; Chevooru SK; Eickelberg O; Hartung HP; Neuhaus O
[Ad] Endereço:Department of Neurology, Heinrich Heine University, Düsseldorf, Germany.
[Ti] Título:Downregulation of monocytic differentiation via modulation of CD147 by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.
[So] Source:PLoS One;12(12):e0189701, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:CD147 is an activation induced glycoprotein that promotes the secretion and activation of matrix metalloproteinases (MMPs) and is upregulated during the differentiation of macrophages. Interestingly, some of the molecular functions of CD147 rely on its glycosylation status: the highly glycosylated forms of CD147 induce MMPs whereas the lowly glycosylated forms inhibit MMP activation. Statins are hydroxy-methylglutaryl coenzyme A reductase inhibitors that block the synthesis of mevalonate, thereby inhibiting all mevalonate-dependent pathways, including isoprenylation, N-glycosylation and cholesterol synthesis. In this study, we investigated the role of statins in the inhibition of macrophage differentiation and the associated process of MMP secretion through modulation of CD147. We observed that differentiation of the human monocytic cell line THP-1 to a macrophage phenotype led to upregulation of CD147 and CD14 and that this effect was inhibited by statins. At the molecular level, statins altered CD147 expression, structure and function by inhibiting isoprenylation and N-glycosylation. In addition, statins induced a shift of CD147 from its highly glycosylated form to its lowly glycosylated form. This shift in N-glycosylation status was accompanied by a decrease in the production and functional activity of MMP-2 and MMP-9. In conclusion, these findings describe a novel molecular mechanism of immune regulation by statins, making them interesting candidates for autoimmune disease therapy.
[Mh] Termos MeSH primário: Basigina/metabolismo
Inibidores de Hidroximetilglutaril-CoA Redutases/química
Receptores de Lipopolissacarídeos/metabolismo
Monócitos/efeitos dos fármacos
[Mh] Termos MeSH secundário: Doenças Autoimunes
Biotinilação
Diferenciação Celular
Membrana Celular/metabolismo
Glicosilação
Seres Humanos
Sistema Imunitário
Macrófagos/citologia
Macrófagos/efeitos dos fármacos
Metaloproteinase 2 da Matriz/metabolismo
Metaloproteinase 9 da Matriz/metabolismo
Monócitos/citologia
Permeabilidade
Fenótipo
Prenilação
Células THP-1
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (BSG protein, human); 0 (Hydroxymethylglutaryl-CoA Reductase Inhibitors); 0 (Lipopolysaccharide Receptors); 136894-56-9 (Basigin); EC 3.4.24.24 (MMP2 protein, human); EC 3.4.24.24 (Matrix Metalloproteinase 2); EC 3.4.24.35 (MMP9 protein, human); EC 3.4.24.35 (Matrix Metalloproteinase 9)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180108
[Lr] Data última revisão:
180108
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171219
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0189701


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[PMID]:29072708
[Au] Autor:Hjuler CT; Maolanon NN; Sauer J; Stougaard J; Thygesen MB; Jensen KJ
[Ad] Endereço:Centre for Carbohydrate Recognition and Signaling, Copenhagen University, Frederiksberg, Denmark.
[Ti] Título:Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies.
[So] Source:Nat Protoc;12(11):2411-2422, 2017 Nov.
[Is] ISSN:1750-2799
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Glycobiology, in particular the study of carbohydrate-protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments. The first stage is to add an oligo(ethylene glycol) linker (OEG-linker) that has a terminal triphenylmethanethiol group to the reducing end of the oligosaccharide by oxime formation catalyzed by aniline. The triphenylmethyl (trityl) tag is then removed from the linker to expose a thiol (stage 2) to allow a conjugation reaction at the thiol group (stage 3). There are many possible conjugation reactions, depending on the desired application. Examples shown in this protocol are as follows: (i) coupling of the oligosaccharide to a support for surface plasmon resonance (SPR) studies, (ii) fluorescence labeling for microscale thermophoresis (MST) or bioimaging, and (iii) biotinylation for biolayer interferometry (BLI) studies. This protocol starts from unprotected carbohydrates and provides glycoconjugates in milligram amounts in just 2 d.
[Mh] Termos MeSH primário: Técnicas de Química Sintética
Glicoconjugados/síntese química
Glicômica/métodos
Lipopolissacarídeos/química
Compostos de Sulfidrila/química
Compostos de Tritil/química
[Mh] Termos MeSH secundário: Compostos de Anilina/química
Biotinilação
Catálise
Interferometria
Imagem Óptica
Oximas/química
Polietilenoglicóis/química
Ligação Proteica
Ressonância de Plasmônio de Superfície
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Aniline Compounds); 0 (Glycoconjugates); 0 (Lipopolysaccharides); 0 (Nod factor IV, Rhizobium meliloti); 0 (Oximes); 0 (Sulfhydryl Compounds); 0 (Trityl Compounds); 30IQX730WE (Polyethylene Glycols); SIR7XX2F1K (aniline)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171102
[Lr] Data última revisão:
171102
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171027
[St] Status:MEDLINE
[do] DOI:10.1038/nprot.2017.109


  9 / 4461 MEDLINE  
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[PMID]:28986262
[Au] Autor:Cheah JS; Yamada S
[Ad] Endereço:Biomedical Engineering Department, University of California, Davis, United States.
[Ti] Título:A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat.
[So] Source:Biochem Biophys Res Commun;493(4):1522-1527, 2017 Dec 02.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis.
[Mh] Termos MeSH primário: Biotina/química
Proteínas/química
Proteínas/isolamento & purificação
[Mh] Termos MeSH secundário: Animais
Biotinilação
Western Blotting
Carbono-Nitrogênio Ligases/genética
Carbono-Nitrogênio Ligases/metabolismo
Cães
Eletroforese em Gel de Poliacrilamida
Proteínas de Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Temperatura Alta
Imunoprecipitação
Células Madin Darby de Rim Canino
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Proteínas Repressoras/genética
Proteínas Repressoras/metabolismo
Solubilidade
Estreptavidina
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Escherichia coli Proteins); 0 (Proteins); 0 (Recombinant Proteins); 0 (Repressor Proteins); 6SO6U10H04 (Biotin); 9013-20-1 (Streptavidin); EC 6.3.- (Carbon-Nitrogen Ligases); EC 6.3.4.15 (birA protein, E coli)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171121
[Lr] Data última revisão:
171121
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171008
[St] Status:MEDLINE


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[PMID]:28973477
[Au] Autor:Einarson OJ; Sen D
[Ad] Endereço:Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
[Ti] Título:Self-biotinylation of DNA G-quadruplexes via intrinsic peroxidase activity.
[So] Source:Nucleic Acids Res;45(17):9813-9822, 2017 Sep 29.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The striking and ubiquitous in vitro affinity between hemin and DNA/RNA G-quadruplexes raises the intriguing possibility of its relevance to biology. To date, no satisfactory experimental framework has been reported for investigating such a possibility. Complexation by G-quadruplexes leads to activation of the bound hemin toward catalysis of 1- and 2-electron oxidative reactions, with phenolic compounds being particularly outstanding substrates. We report here a strategy for exploiting that intrinsic peroxidase activity of hemin•G-quadruplex complexes for self-biotinylation of their G-quadruplex component. Such self-biotinylation occurs with good efficiency and high discrimination in vitro, being specific for G-quadruplexes and not for duplexes. The biotinylated DNA, moreover, remains amenable to polymerase chain reaction amplification, rendering it suitable for analysis by ChIP-Seq and related methods. We anticipate that this self-biotinylation methodology will also serve as a sensitive tool, orthogonal to existing ones, for identifying, labeling and pulling down cellular RNA and DNA G-quadruplexes in general, as well as proteins bound to or proximal to such quadruplexes.
[Mh] Termos MeSH primário: DNA Catalítico/química
Quadruplex G
Hemina/química
Oligonucleotídeos/química
Peroxidases/química
[Mh] Termos MeSH secundário: Biocatálise
Técnicas Biossensoriais/métodos
Biotina/química
Biotinilação
Peróxido de Hidrogênio/química
Cinética
Mimetismo Molecular
Oxirredução
Fenóis/química
Reação em Cadeia da Polimerase
Estreptavidina/química
Tiramina/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Catalytic); 0 (Oligonucleotides); 0 (Phenols); 0 (biotin tyramine); 6SO6U10H04 (Biotin); 743LRP9S7N (Hemin); 9013-20-1 (Streptavidin); BBX060AN9V (Hydrogen Peroxide); EC 1.11.1.- (Peroxidases); X8ZC7V0OX3 (Tyramine)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171017
[Lr] Data última revisão:
171017
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171004
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx765



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