Desenvolvimento do método NU-ELISA foi apoiado pelo NIH conceder RO1AG023687.

1. Cultura de células de mamíferos NU-ELISA pode ser realizado em qualquer tipo de células de mamíferos que podem ser cultivadas em cultura. Nós preferimos preparar moderada a grandes lotes de células para que nucleossomos podem ser isoladas em quantidade suficiente para preparar vários pratos idênticos carregados ELISA, permitindo assim que os testes com vários anticorpos diferentes (Abs). As escalas cultura a seguir fornecem amplo material: De células-tronco do mouse embyronic, crescer um ou dois pratos de 15 cm de células sem células alimentadoras. Para fibroblastos, crescer 5-10 cm para 15 placas de confluência. 2. Isolamento de Núcleos Nota: Todos os passos estão no gelo com pré-refrigerados buffers, excepto quando indicado. <ol><li> Trypsinize células com 3 ml de tripsina por placa, combinar e adicionar 20 ml de PBS gelado / butirato. Centrifugar a 1000 rpm por 5 min. </li><li> Ressuspender as células em 10 ml PBS / butirato e centrifugar a 1000 rpm por 5 min. </li><li> Ressuspender em 4 ml tampão de lise com inibidores da protease (Sigma-Aldrich P-8340). Dounce homogeneizar 20 cursos com pilão tipo B no gelo. </li><li> Centrifugar a 2000g por 10 min a 4 ° C. (O sobrenadante contém citoplasma, o que não é necessário para este protocolo, mas pode ser economizada para outros usos, se desejar). </li><li> Ressuspender o sedimento em 2 ml de gelo frio tampão de lavagem C (com inibidores da protease). </li><li> Camada do material ressuspenso mais de 5 ml almofada sacarose 30%, então centrifugue a 2400g por 5 min em um rotor de caçamba móvel. Núcleos migram através almofada, e os restos permanece na interface. </li><li> Retire todo o volume de líquido com cuidado e ressuspender os núcleos em 250 mL de tampão de lavagem gelada C + inibidores da protease. </li></ol> 3. Isolamento de Nucleossomos por in situ Micrococcal Nuclease Digestão (MNase) Usamos um procedimento em que a cromatina é digerida in situ dentro de núcleos, infundindo-lhes MNase, seguido por um tratamento hipotônico para livre no disco nucleossomos intactos no sobrenadante. <ol><li> Adicionar 3 mL 0,1 M CaCl 2 para a amostra. Coloque em 37 ° C em bloco de calor. Permitem assumir 37 ° C de temperatura. </li><li> Adicione 2 unidades MNase (Micrococcal Nuclease, Sigma-Aldrich, dissolvido em 2 units/10μl em tampão MNase), então incubar a 37 ° C por 12 min, com mistura freqüentes usando a ponta da pipeta. </li><li> Adicionar 6 mL de 0,5 M pH Na-EDTA, 8,0 para parar a reação. Coloque sobre o gelo. </li><li> Centrifugar a 2000g por 4 min, elimine o sobrenadante. Ressuspender o sedimento em 300 mL 0,2 mM Na-EDTA. Loja em gelo por 1 h com pipetagem suave ocasionais. (Estas condições hipotônica libertar nucleossomos livre de núcleos). </li><li> Centrifugar a 3000 g por 4 min a 4 ° C. Salve o sobrenadante, que contém nucleossomos livre, no gelo. </li><li> Ressuspender o sedimento novamente com 300 mM 0,2 mL de Na-EDTA. Loja em gelo por 1 h com pipetagem suave ocasionais. </li><li> Centrifugar a 3000g por 4min a 4 ° C. Manter o sobrenadante e combinar com o primeiro sobrenadante da etapa 5. Os preparativos resultando mononucleosome pode ser aliquotado e estocado a -80 ° C. </li></ol> Nota: A quantidade de cromatina pode ser grosseiramente quantificada pela medição da absorvência a 260 nm de uma amostra de 10 ml adicionadas a 990 mL de água. A 260 = 10 (após o ajuste para a diluição 1 / 100) corresponde a cerca de 1mg/ml de cromatina. Além disso, durante o procedimento acima, pequenas amostras podem ser retidos e posteriormente analisadas por seu conteúdo de DNA para monitorar a qualidade ea extensão da digestão MNase, que deve conter predominantemente DNA de 146 bp de comprimento. Laddering é indicativo de digestão MNase incompleta. Nota: Fig.1 contém um resumo diagramado de isolamento nuclear e as etapas de digestão MNase. 4. Nucleossomo-ELISA (NU-ELISA) Detectamos PTMs em frações contendo nativa intacta nuclesosomes que foram imobilizados em 96 placas de ELISA bem microtitulação. Para cada amostra, fazemos uma série de duas diluições, e estes são revestidos em poços em triplicado. <ol><li> Revestir as placas Maxisorp overnight a 4 ° C com 50 ul / poço de nucleossomos diluída em tampão de revestimento. Sugeriu diluições de série do nucleossomos são preparados a partir de linha superior a linha de fundo pela adição de 0,1 mg, 0,05 mg, 0,025 mg, 0,0125 g, 0,00625 mg, 0,00313 mg, 0,00156 mg, com tampão de revestimento apenas (0 cromatina mg) no fundo linha. (Nota: cobre placa são necessários para esta etapa.) </li><li> De manhã, lave os pratos 4 vezes com 200 mL / poço PBS/0.5% Tween-20 em temperatura ambiente (RT) para um total combinado de 10 min com uma máquina de lavar prato. </li><li> Bloco 1 hora em temperatura ambiente com 100 ul / poço PBS/0.05% BSA Tween-20% / 5. </li><li> Remova o tampão de bloqueio, agitando a placa invertida rapidamentesobre uma pia. Cubra e guarde as placas a -20 ° C, ou ir diretamente para a próxima etapa. </li><li> Adicionar 1 ° Abs em um volume de 50 l / bem diluído 1:1000 (ou conforme necessário) em PBS/0.05% Tween-20% / 5 BSA, incubar a RT por 1 hora em um rotador. </li><li> Lavar as placas 4 vezes com 200 mL / poço PBS/0.5% Tween-20 em RT e para um total combinado de 10 min em uma máquina de lavar prato. </li><li> Adicionar peroxidase de rábano (HRP) conjugado com 2 ° Abs, diluído 1:5000 (ou quando necessário) em PBS/0.05% Tween-20% / 5 BSA em temperatura ambiente por uma hora em um rotador. </li><li> Lavar as placas 4 vezes com 200 mL / poço PBS/0.5% Tween-20. Cada lavagem é de RT e para um total de 10 minutos com uma máquina de lavar prato. </li><li> Desenvolver as placas, adicionando 50 ul de substrato TMB em cada poço por 10 min em temperatura ambiente. Pare a reação adicionando 50 ul / poço de 2N H 2 SO 4 e ler as placas de 450 nm. (Dica: Centrifugar a 1500 rpm por 2 min usando uma placa de rotor para dissipar as bolhas de ar nos poços antes absorbâncias leitura). </li><li> Export as leituras de arquivos de planilhas para análises quantitativas e estatísticas. </li></ol> Reagentes <table border="1"><tbody><tr><td> PBS / butirato </td></tr><tr><td> 135 mM NaCl </td></tr><tr><td> 2,5 mM KCl </td></tr><tr><td> 8 mM Na 2 HPO 4 </td></tr><tr><td> 1,5 mM KH 2 PO 4 </td></tr><tr><td> 10 mM Na-butirato </td></tr></tbody></table><table border="1"><tbody><tr><td> Tampão de Lise </td></tr><tr><td> 250 mM de sacarose </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM Na-butirato </td></tr><tr><td> 4 mM MgCl 2 </td></tr><tr><td> 0,1% Triton X-100 </td></tr></tbody></table><table border="1"><tbody><tr><td> Tampão de lavagem C </td></tr><tr><td> 250 mM de sacarose </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM Na-butirato </td></tr><tr><td> 4 mM MgCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Almofada sacarose </td></tr><tr><td> 30% (w / v) sacarose em tampão de lavagem C </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal Nuclease buffer </td></tr><tr><td> 5 mM NaPO 4 tampão, pH 7,0 </td></tr><tr><td> 0,025 mM CaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Buffer de revestimento </td></tr><tr><td> Solução 80ml A + B + Solução 170ml 250ml dH 2 O </td></tr><tr><td> A solução: 0,2 M Na 2 CO 3 </td></tr><tr><td> Solução B: 0,2 M NaHCO 3 </td></tr></tbody></table> 5. Resultados representante Utilizando o método ELISA-NU, uma série de várias placas idênticas são preparados que podem ser carregados com cromatina preparada na forma de mononucleosomes. Normalmente, prepare série de idêntica-carregado placas de modo que cada um pode ser sondado com diferentes anti-PTM anticorpos específicos (Abs). Nós também sempre preparar um prato que pode ser sondado com uma Ab que detecta histonas sem levar em conta a sua alteração de controle de carga total do Estado de cromatina. Este controle é essencial para depois comparar quantitativamente o conteúdo PTM de nucleossomos preparados a partir de amostras diferentes. Para quantificar os níveis de PTMs dentro de uma amostra cromatina dado que corrigir os valores de absorbância primas usando esta abordagem: Primeiro, nós subtrair qualquer sinal de fundo usando os valores obtidos a partir de poços de controlo que não contém cromatina (fundo é geralmente insignificante). Em seguida, padronizar PTM específicos sinais para NU-ELISA resultados obtidos a partir de uma placa idêntica carregado que foi ensaiada com Ab que detecta nucleossomos independente de seu estado de modificação. (Nós usamos Abs policlonal específico para histonas H2A ou H2B para esta finalidade). Nós, então, determinar médias e variâncias para cada diluição da cromatina, e utilizar os dados obtidos a partir da porção linear do ensaio ELISA. Exemplos detalhados de NU-ELISA análises matemáticas e estatísticas têm sido relatados anteriormente 4 <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="Figura 1"/> Figura 1. Esquemática diagam de NU-ELISA passos. Células A. mamíferos são colhidas; Núcleos B. são separados a partir de células por Dounce homogenization, C. Disrupted células são carregadas em cima de uma almofada de sacarose 30% w / v, D. Após a centrifugação, os núcleos são depositados no sedimento; E, F. Chromatin são digeridos in situ para mononucleomes predominantemente por MNase; G, H , eu. mononucleosomes solúveis são extraídos por tratamento hipotônico e centrifugação repetida de material nuclear residual. Mononucleosomes J. a partir de amostras diferentes (rotulado samp. 1 e 2, neste caso) são revestidos em poços da microplaca em série de forma idêntica-carregado com placas e interrogado PTM específicos Abs e PTM independente Abs para determinar o carregamento cromatina total. K. Representação dos níveis de diferencial sinal de detecção em duas amostras que diferem em seu conteúdo PTM, mas tem conteúdo cromatina global semelhante, como julgado por anti-H2B imunorreatividade.

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

Não há conflitos de interesse declarados.

NU-ELISA fornece um método para determinar o status global de PTMs nucleosome presente dentro de um tipo particular de célula. NU-ELISA estudos têm mostrado que estados globais modificação nucleosome diferem em comparações de tipos de células diferentes 4. Além disso, os perfis de NU-ELISA PTM mudar quando as células são expostas a agentes moduladores epigenéticas, como tricostatina-A ou quando as células-tronco do mouse embyronic são diferenciadas 4. O método também tem sido aplicada com sucesso para estudar a cromatina de células-tronco embrionárias humanas 5. É importante notar a NU-ELISA, na sua forma actual, pode detectar apenas a assinatura do composto de PTMs presentes na soma total do epigenoma celular. Isso difere marcadamente de genome-wide imunoprecipitação da cromatina, que pode determinar a distribuição de um PTM específicos através específicas loci genéticos. Os passos iniciais dos procedimentos NU-ELISA são adaptados a partir de métodos anteriores para isolar mononucleosomes de núcleos de mamíferos 6,7. Estes métodos adaptados fornecem uma maneira conveniente de obter alta qualidade mononucleosomes intacta a partir de células de mamíferos, e as frações resultantes são abrangentes em seu conteúdo cromatina, mas contêm uma grande quantidade de material nuclear adicional. Desde Abs específicos são usados, contaminando não nucleosomal material é bem tolerado no ensaio NU-ELISA, ao contrário de massa-spec abordagens que exigem uma maior pureza. No entanto, NU-ELISA é mais sensível e quantitativa de western blotting 4, proporcionando assim uma boa alternativa aos westerns e especificação de massa para a detecção de PTMs nucleosomal.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Micrococcal Nucleae </td><td> Sigma </td><td> N3755 </td><td> Faça alíquotas de buffer de 2 U/10μl, armazenadas a -20 ° C </td></tr><tr><td> Nunc Maxisorp placas de microtitulação </td><td> Pescador </td><td> 12-565-135 </td><td> Cobre placa adesiva também podem ser encomendados através Fisher </td></tr><tr><td> Louça Placa automatizado </td><td></td><td></td><td></td></tr><tr><td> 1 Passo-substrato TMB-ELISA </td><td> Perfurar </td><td> 34028 </td><td> Armazenar a 4 ° C pré-aquecido a RT antes da utilização ou conforme indicado na folha de especificações </td></tr><tr><td> Placa de microtitulação Leitor </td><td> Bio-Rad leitora de microplacas (modelo: 680) </td><td></td><td> A maioria dos modelos leitor colorimétrico placa são adequados </td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

O genoma dos eucariotos existe como cromatina, que contém DNA e proteínas. A unidade fundamental da cromatina é o nucleossomo, que contém 146 pares de bases de DNA associados a cada uma das duas histonas H2A, H2B, H3, H4 e 1. As caudas N-terminal de histonas são ricas em lisina e arginina e são modificados pós-transcricionalmente por acetilação, metilação e outras modificações pós-translacionais (PTMs). A configuração PTM de nucleossomos podem afetar a atividade transcricional de DNA associados, proporcionando assim um modo de regulação do gene que é epigenética na natureza 2,3. Nós desenvolvemos um método chamado nucleosome ELISA (NU-ELISA) para determinar quantitativamente assinaturas PTM global de nucleossomos extraído de células. NU-ELISA é mais sensível e quantitativa de western blotting, e é útil para interrogar o estado epiproteomic de tipos específicos de células. Este artigo de jornal vídeo mostra os procedimentos detalhados para realizar NU-ELISA análise.

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Nucleossomo ELISA (NU-ELISA) é um método sensível e quantitativa para detectar padrões globais de modificações pós-translacionais em preparações de natal, nucleossomos intactos. Essas modificações incluem methylations, acetylations e fosforilações em específico histona resíduos de aminoácidos e, portanto, NU-ELISA fornece um ensaio global de proteômica do total estados de modificação da cromatina de tipos específicos de células.

Detecção de modificações pós-translacionais sobre Native Nucleossomos Intact por ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

detection-post-translational-modifications-on-native-intact

Research

JoVE Journal

Biology

17.2K visualizações.  Stanford University. Nucleossomo ELISA (NU-ELISA) é um método sensível e quantitativa para detectar padrões globais de modificações pós-translacionais em preparações de natal, nucleossomos intactos. Essas modificações incluem methylations, acetylations e fosforilações em específico histona resíduos de aminoácidos e, portanto, NU-ELISA fornece um ensaio global de proteômica do total estados de modificação da cromatina de tipos específicos de células.

Vídeo: Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Detecção de Metaloproteinases da Matriz Funcional por zimografia

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Biologia

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analisando a função de GTPases Pequenas pela microinjeção de Plasmids em Polarized Células Epiteliais

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Detecção de histona Modificações nas folhas das plantas

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Brotando Yeast extração e purificação de proteínas para o estudo da função, interações e modificações pós-traducionais

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificação de modificações pós-translacionais de Complexos de proteína vegetal

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Face Detection En de óxido nítrico e superóxido na camada endotelial das artérias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimento CRISPR Vector construção e caracterização de DNA Modificações pela geração de raízes de tomateiro Cabeludo

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Fluxo de trabalho completo para Análise de histona modificações pós-traducionais Usando Bottom-up Espectrometria de Massa: De histona Extração de Análise de Dados

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Comparação de tabaco métodos de remoção de células hospedeiras de proteínas por descascamento plantas intactas ou por tratamento térmico de extratos

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...