Développement de la méthode ELISA-NU a été soutenue par NIH RO1AG023687.

1. Culture de cellules mammaliennes NU-ELISA peut être effectuée sur n&#39;importe quel type de cellules de mammifères qui peuvent être mises en culture. Nous préférons pour préparer modéré à d&#39;importants lots de cellules de sorte que les nucléosomes peuvent être isolés en quantité suffisante pour préparer plusieurs identique chargés plaques ELISA, permettant ainsi des tests avec plusieurs anticorps différents (Abs). Les échelles de culture suivants fournissent un abondant matériel: Pour les cellules de souris tige embyronic, pousser un ou deux plaques 15 cm de cellules sans cellules nourricières. Pour les fibroblastes, croissance de 5 à 10 15 cm à plaques confluent. 2. Isolement des noyaux Remarque: Toutes les étapes sont sur la glace avec pré-réfrigérée tampons, sauf comme indiqué. <ol><li> Trypsiniser cellules avec 3 ml de trypsine par assiette, mélanger, et ajouter 20 ml de PBS glacé / butyrate. Centrifuger à 1000 rpm pendant 5 min. </li><li> Resuspendre les cellules dans 10 ml de PBS / butyrate et centrifuger à 1000 rpm pendant 5 min. </li><li> Remettre en suspension dans 4 ml avec un tampon de lyse inhibiteurs de la protéase (Sigma-Aldrich P-8340). Dounce homogénéiser les 20 coups de pilon de type B sur la glace. </li><li> Centrifuger à 2000g pendant 10 min à 4 ° C. (Le surnageant contient cytoplasme, ce qui n&#39;est pas nécessaire pour ce protocole, mais il peut être sauvegardé pour d&#39;autres usages si désiré). </li><li> Reprendre le culot dans 2 ml de glace froide tampon de lavage C (avec des inhibiteurs de la protéase). </li><li> Couche le matériel remis en suspension de plus de 5 ml coussin de saccharose à 30%, puis centrifuger à 2400g pendant 5 min dans un rotor swing. Les noyaux vont migrer à travers coussin, et les débris restent à l&#39;interface. </li><li> Retirez tout le volume de liquide soigneusement et remettre en suspension les noyaux de glace de 250 tampons de lavage à froid ul C + inhibiteurs de protéase. </li></ol> 3. Isolement des nucléosomes in situ par des Micrococcal nucléase (MNase) Digestion Nous utilisons une procédure dans laquelle la chromatine est digérée in situ au sein des noyaux en leur insufflant MNase, suivie d&#39;un traitement hypotonique à conduire sans nucléosomes intacte dans le surnageant. <ol><li> Ajouter 3 pi à 0,1 M CaCl 2 à l&#39;échantillon. Mettre en bloc de 37 ° C chaleur. Permettez à assumer 37 ° C de température. </li><li> Ajouter 2 unités MNase (Micrococcal nucléase, Sigma-Aldrich, dissous à 2 dans un tampon de units/10μl MNase), puis incuber pendant à 37 ° C pendant 12 minutes, en mélangeant souvent avec une pointe de pipette. </li><li> Ajouter 6 ul de 0,5 M Na-EDTA, pH 8,0 pour arrêter la réaction. Mis sur la glace. </li><li> Centrifuger à 2000g pendant 4 min, éliminer le surnageant. Reprendre le culot dans 300 pl 0,2 mM Na-EDTA. Store sur la glace pendant 1 h avec pipetage doux occasionnels. (Ces conditions hypotoniques libérer nucléosomes libres de noyaux). </li><li> Centrifuger à 3000 g pendant 4 min à 4 ° C. Enregistrer le surnageant, qui contient des nucléosomes libres, sur la glace. </li><li> Reprendre le culot à nouveau avec 300 pl 0,2 mM Na-EDTA. Store sur la glace pendant 1 h avec pipetage doux occasionnels. </li><li> Centrifuger à 3000g pour 4min à 4 ° C. Conservez le surnageant et de combiner avec le premier surnageant de l&#39;étape 5. Les préparatifs résultant mononucleosome peut être aliquotés et conservés à -80 ° C. </li></ol> Remarque: Le montant de la chromatine peuvent être grossièrement quantifiée en mesurant l&#39;absorbance à 260 nm d&#39;un échantillon de 10 ul ajouté à 990 pl d&#39;eau. A 260 = 10 (après ajustement pour la dilution 1 / 100) correspond à environ 1mg/ml de la chromatine. En outre, durant la procédure ci-dessus, de petits échantillons peuvent être conservés et, plus tard analysés pour leur contenu en ADN pour contrôler la qualité et l&#39;étendue des digestions MNase, qui devrait surtout contenir de l&#39;ADN de 146 pb de longueur. L&#39;échelonnement est indicatif de la digestion MNase incomplète. Remarque: Fig.1 contient un résumé schématisés d&#39;isolement nucléaire et les digestions MNase étapes. 4. Nucléosome-ELISA (NU-ELISA) Nous détectons PTM sur les fractions contenant des espèces autochtones nuclesosomes intacts qui ont été immobilisés sur des plaques de microtitration de 96 puits ELISA. Pour chaque échantillon, nous faisons une série de deux dilutions, et elles sont appliquées sur des puits en triple exemplaire. <ol><li> Déposer sur des plaques MaxiSorp nuit à 4 ° C avec 50 ul / puits de nucléosomes dilués dans un tampon de revêtement. Suggestions de dilutions en série de nucléosomes sont préparés à partir de rangée du haut de la rangée du bas en ajoutant 0,1 mg, 0,05 mg, 0,025 mg, 0,0125 g, 0,00625 mg, 0,00313 mg, 0,00156 mg, avec le tampon seul revêtement (0 chromatine mg) dans le fond rangée. (Note: couvre la plaque sont nécessaires pour cette étape.) </li><li> Dans la matinée, se laver les plaques 4 fois avec 200 pl / puits PBS/0.5% Tween-20 à température ambiante (TA) pour un total combiné de 10 min avec un laveur de plaques. </li><li> Bloc 1 h à température ambiante avec 100 ul / puits PBS/0.05% de Tween-20 / 5% de BSA. </li><li> Retirez le tampon de blocage en secouant la plaque inversée brusquementdessus d&#39;un évier. Couvrez et conservez les plaques à -20 ° C, ou aller directement à l&#39;étape suivante. </li><li> Ajouter 1 ° Abs dans un volume de 50 pl / puits dilué 1:1000 (ou au besoin) dans PBS/0.05% de Tween-20 / 5% de BSA, incuber à température ambiante pendant 1 heure sur un agitateur. </li><li> Laver les plaques 4 fois avec 200 pl / puits PBS/0.5% Tween-20 à température ambiante et pour un total combiné de 10 min dans un laveur de plaques. </li><li> Ajouter la peroxydase de raifort (HRP) conjuguée à 2 ° Abs, dilué 1:5000 (ou au besoin) dans PBS/0.05% de Tween-20 / 5% de BSA à température ambiante pendant une heure sur un agitateur. </li><li> Laver les plaques 4 fois avec 200 pl / puits PBS/0.5% de Tween-20. Chaque lavage est à température ambiante et pour un total de 10 minutes avec un laveur de plaques. </li><li> Développer les plaques en ajoutant 50 pl de substrat TMB dans chaque puits pendant 10 min à température ambiante. Arrêter la réaction en ajoutant 50 pl / puits de 2N H 2 SO 4 et de lire les plaques de 450 nm. (Astuce: Centrifuger à 1500 rpm pendant 2 min à l&#39;aide d&#39;un rotor plaque à dissiper les bulles d&#39;air dans le puits avant la lecture absorbances). </li><li> Exporter les lectures de fichiers tableur pour des analyses quantitatives et statistiques. </li></ol> Réactifs <table border="1"><tbody><tr><td> PBS / butyrate </td></tr><tr><td> 135 mM de NaCl </td></tr><tr><td> 2,5 mM de 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 de Na-butyrate </td></tr></tbody></table><table border="1"><tbody><tr><td> Lysis Buffer </td></tr><tr><td> 250 mM de saccharose </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM de Na-butyrate </td></tr><tr><td> 4 mM de MgCl2 </td></tr><tr><td> 0,1% de Triton X-100 </td></tr></tbody></table><table border="1"><tbody><tr><td> Laver le tampon C </td></tr><tr><td> 250 mM de saccharose </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM de Na-butyrate </td></tr><tr><td> 4 mM de MgCl2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Coussin de saccharose </td></tr><tr><td> 30% (p / v) de saccharose dans le tampon de lavage C </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal nucléase Tampon </td></tr><tr><td> 5 mM NaPO 4 tampon, pH 7,0 </td></tr><tr><td> 0,025 mM CaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Tampon de revêtement </td></tr><tr><td> Solution 80ml 170ml Solution A + B + 250ml dH 2 O </td></tr><tr><td> Solution A: 0,2 M Na 2 CO 3 </td></tr><tr><td> Solution B: 0,2 M de NaHCO 3 </td></tr></tbody></table> 5. Les résultats représentatifs En utilisant la méthode ELISA-NU, une série de plusieurs plaques identiques sont préparés qui peuvent être chargés avec la chromatine préparées sous la forme d&#39;mononucleosomes. Nous préparent habituellement série de plaques à l&#39;identique-chargés afin que chacun peut être sondé avec différents anti-PTM anticorps spécifiques (Abs). Nous avons également toujours préparer une plaque qui peut être sondé avec un Ab qui détecte les histones, sans égard à leur état de modification de chargement chromatine contrôle total. Ce contrôle est indispensable pour pouvoir ensuite comparer quantitativement le contenu du PTM nucléosomes préparés à partir d&#39;échantillons différents. Pour quantifier les niveaux de PTM dans un échantillon donné, nous chromatine corriger les valeurs d&#39;absorbance brutes en utilisant cette approche: d&#39;abord, nous soustrayons tout signal de fond en utilisant les valeurs obtenues à partir de puits de contrôle ne contenant pas de chromatine (fond est généralement négligeable). Nous avons ensuite normaliser PTM signaux spécifiques aux NU-ELISA résultats obtenus à partir d&#39;une plaque identique chargé qui a été dosé avec un Ab qui détecte nucléosomes indépendamment de leur état de modification. (Nous avons utilisé des anticorps polyclonaux dirigés spécifiques des histones H2A ou H2B à cet effet). Nous avons ensuite déterminer les moyens et les écarts pour chaque dilution de la chromatine, et utiliser les données obtenues à partir de la portion linéaire du test ELISA. Des exemples détaillés de NU-ELISA analyses mathématiques et statistiques ont été rapportés précédemment 4 <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="Figure 1"/> Figure 1. Schéma Diagam des NU-ELISA étapes. A. Cellules mammifères sont récoltés; Noyaux B. sont séparés de cellules par Dounce homogenization, C. Perturbation cellules sont chargées sur le dessus d&#39;un coussin de saccharose p / v 30%, D. Après centrifugation, les noyaux sont déposés dans le culot; E, F. chromatine sont digérées in situ à prédominance mononucleomes par MNase; G, H , je. mononucleosomes solubles sont extraites par un traitement hypotonique et centrifugation répétée de matières nucléaires résiduelles. Mononucleosomes juge à partir d&#39;échantillons différents (SAMP étiquetés. 1 et 2 dans ce cas) sont enduits sur des puits de microtitration en série de manière identique-chargé avec des plaques et interrogé Le PTM-spécifiques Abs et PTM-Abs indépendante pour déterminer le chargement chromatine totale. Représentation des niveaux K. signal différentiel de détection dans deux échantillons qui diffèrent dans leur contenu PTM, mais ont les mêmes contenus chromatine globale, à en juger par les anti-H2B immunoréactivité.

<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>

NU-ELISA fournit une méthode pour déterminer l&#39;état mondial des PTM nucléosome présents dans un type cellulaire particulier. NU-ELISA études ont montré que mondiale états de modification du nucléosome diffèrent dans les comparaisons de types de cellules divergentes 4. En outre, les NU-ELISA profils PTM changer lorsque les cellules sont exposées à des agents modulateurs épigénétiques comme trichostatine-A ou lorsque les cellules de souris tige embyronic sont différenciées 4. La méthode a également été appliquée avec succès pour étudier la chromatine des cellules ES humaines 5. Il est important de noter la NU-ELISA, dans sa forme actuelle, ne peuvent détecter la signature composite de PTM présents dans la somme totale de l&#39;épigénome cellulaire. Cela diffère nettement de l&#39;ensemble du génome immunoprécipitation de la chromatine, qui peut déterminer la distribution d&#39;un PTM spécifiques à travers des loci génétiques spécifiques. Les étapes initiales de la procédure NU-ELISA sont adaptés à partir des méthodes précédentes pour isoler mononucleosomes à partir des noyaux de mammifères 6,7. Ces méthodes adaptées fournissent un moyen rapide d&#39;obtenir de haute qualité mononucleosomes intacts de cellules de mammifères, et les fractions obtenues sont complètes dans leur contenu de la chromatine, mais contiennent beaucoup de matières nucléaires supplémentaires. Depuis Abs spécifiques sont utilisées, contaminent non nucléosomiques matériel est bien toléré dans le test ELISA-NU, à la différence de masse-spec approches qui nécessitent une plus grande pureté. Toutefois, NU-ELISA est plus sensible et plus quantitative que western blot 4, offrant ainsi de bonnes alternatives à des westerns et spectrométrie de masse pour la détection de PTM nucléosomes.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Micrococcal Nucleae </td><td> Sigma </td><td> N3755 </td><td> Faire des aliquotes de 2 tampons U/10μl, conservés à -20 ° C </td></tr><tr><td> Nunc MaxiSorp microplaques </td><td> Fisher </td><td> 12-565-135 </td><td> Couvre la plaque adhésive peut également être commandé auprès de Fisher </td></tr><tr><td> Laveuse automatique des plaques </td><td></td><td></td><td></td></tr><tr><td> 1-Step TMB-substrat ELISA </td><td> Pierce </td><td> 34028 </td><td> Conserver à 4 ° C préchauffer à température ambiante avant l&#39;utilisation ou tel qu&#39;indiqué sur la fiche technique </td></tr><tr><td> Microtitration lecteur de plaque </td><td> Bio-Rad lecteur de microplaques (modèle: 680) </td><td></td><td> La plupart des modèles colorimétriques lecteur de plaque sont adaptés </td></tr></tbody></table>

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

Le génome des eucaryotes existe comme la chromatine, qui contient à la fois l&#39;ADN et des protéines. L&#39;unité fondamentale de la chromatine est le nucléosome, qui contient 146 paires de bases d&#39;ADN associé à deux chacune des histones H2A, H2B, H3 et H4 1. Les queues N-terminale des histones sont riches en lysine et l&#39;arginine et sont modifiés après-transcriptionnellement par acétylation, la méthylation, et d&#39;autres modifications post-traductionnelles (PTM). La configuration du PTM nucléosomes peuvent affecter l&#39;activité transcriptionnelle de l&#39;ADN associées, fournissant ainsi un mode de régulation des gènes qui sont de nature épigénétique 2,3. Nous avons développé une méthode appelée nucléosome ELISA (NU-ELISA) pour déterminer quantitativement les signatures PTM mondial de nucléosomes extrait des cellules. NU-ELISA est plus sensible et plus quantitative que western blot, et il est utile d&#39;interroger l&#39;état epiproteomic de types cellulaires spécifiques. Cet article de journal vidéo montre des procédures détaillées pour effectuer les NU-ELISA analyse.

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

Nucléosomes ELISA (NU-ELISA) est une méthode sensible et quantitative pour détecter les tendances mondiales de l&#39;modifications post-traductionnelles dans les préparations de maternelle, nucléosomes intacte. Ces modifications incluent méthylations, acétylations, et des phosphorylations au histones spécifiques résidus d&#39;acides aminés, et donc NU-ELISA fournit une analyse globale de protéomique de l&#39;ensemble, les États modification de la chromatine des types cellulaires spécifiques.

Détection des modifications post-traductionnelles sur les autochtones nucléosomes Intact par 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 ...

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Biology

17.2K vues.  Stanford University. Nucléosomes ELISA (NU-ELISA) est une méthode sensible et quantitative pour détecter les tendances mondiales de l'modifications post-traductionnelles dans les préparations de maternelle, nucléosomes intacte. Ces modifications incluent méthylations, acétylations, et des phosphorylations au histones spécifiques résidus d'acides aminés, et donc NU-ELISA fournit une analyse globale de protéomique de l'ensemble, les États modification de la chromatine de...

Vidéo: 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.

Détection des métalloprotéinases matricielles fonctionnelle par zymographie

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

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Biologie

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.

Analyse de la fonction des petites GTPases par micro-injection de plasmides dans les cellules épithéliales polarisées

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.

Détection des modifications des histones dans les feuilles des plantes

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.

Bourgeonnement de levure extraction et purification des protéines pour l&#39;étude de la fonction, Interactions, et des modifications post-traductionnelles

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.

Identification des modifications post-traductionnelles des protéines végétales Complexes

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.

Isolement des régions génomiques spécifiques et l&#39;identification des molécules associées par 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.

En détection de visage de l&#39;oxyde nitrique et de superoxyde dans la couche endothéliale des artères intactes

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.

Haut-débit CRISPR Vector Construction et caractérisation de l&#39;ADN Modifications par génération de tomate Poilu Roots

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.

Flux de travail complet pour l&#39;analyse des histones modifications post-traductionnelles utilisant la spectrométrie de masse Bottom-up: De Histone Extraction à l&#39;analyse des données

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.

Comparaison de tabac Méthodes hôte cellulaire Protein suppression par Blanchir plantes intactes ou par traitement thermique des extraits

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