Name: quantitative analysis of chromatin proteomes in disease
Uploaded: 2012-12-28T12:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Chromatin Proteomes in Disease at JoVE.com

Le laboratoire Vondriska est financée par des subventions de l&#39;Institut National Heart, Lung and Blood du NIH et la Fondation Laubisch à l&#39;UCLA. EM est destinataire de la Jennifer S. Buchwald bourse d&#39;études supérieures en physiologie à l&#39;UCLA; HC est le destinataire d&#39;un American Heart Association pré-doctoral, le député est le récipiendaire d&#39;une NIH Ruth Kirschstein post-doctoral, et SF est le récipiendaire du une NIH K99 Award.

Le flux de travail expérimental comporte sept étapes principales (figure 1). Pour tout travail impliquant des échantillons qui seront exécutés sur le spectromètre de masse, l&#39;expérimentateur doit porter une blouse, des gants et un filet à cheveux et prendre soin d&#39;éviter la contamination par la poussière et la chute personnelle de la kératine. 1. Homogénéisation coeur et isolement nucléaire Coeurs de souris sont homogénéisé...

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Deux principales méthodes d&#39;isolement nucléaire ont été examinés précédemment: 27 on est la technique d&#39;homogénéisation Behrens tissu lyophilisé dans un solvant non aqueux et le second, une modification, dont nous utilisons ici, d&#39;homogénéisation des tissus dans une solution aqueuse de saccharose / solution saline suivie par différence de densité ou à gradient de centrifugation. Sous-fractionnement des noyaux par extraction à l&#39;acide sur des échant...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Dulbeco Modified Eagle Medium </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> Protéase à granulés </td><td> Roche </td><td> 04 693 159 001 </td></tr><tr><td> Tamis de 100 um </td><td> BD Falcon </td><td> 352360 </td></tr><tr><td> Ultracut ultramicrotome </td><td> Reichert </td><td></td></tr><tr><td> 100CX électronique à transmission Microscope </td><td> JEOL USA, Inc </td><td></td></tr><tr><td> Loriot </td><td> BioRad </td><td> 161-0496 </td></tr><tr><td> Histone H2A anticorps </td><td> Santa Cruz </td><td> sc-8648 </td></tr><tr><td> Nucléoporine p62 anticorps </td><td> BD Biosciences </td><td> 610498 </td></tr><tr><td> Adénine nucléoanticorps transporteur marée </td><td> Santa Cruz </td><td> sc-9299 </td></tr><tr><td> BiP anticorps </td><td> Santa Cruz </td><td> sc-1050 </td></tr><tr><td> Anticorps tubuline </td><td> Sigma </td><td> T1568 </td></tr><tr><td> Histone H3 anticorps </td><td> Abcam </td><td> ab1791 </td></tr><tr><td> Anticorps fibrillarine </td><td> Signalisation cellulaire </td><td> C12C3 </td></tr><tr><td> SNRP70 anticorps </td><td> Abcam </td><td> ab51266 </td></tr><tr><td> E2F-1 anticorps </td><td> Thermo Fisher </td><td> MS-879 </td></tr><tr><td> Anticorps rétinoblastome </td><td> BD Biosciences </td><td> 554136 </td></tr><tr><td> Factor-1 inductible par l&#39;hypoxie anticorps </td><td> Novus Biologicals </td><td> NB100-469 </td></tr><tr><td> Dosage de protéines BCA </td><td> Thermo Scientific </td><td> 23227 </td></tr><tr><td> Colonne en phase inverse </td><td> Obj Nouveaucace </td><td> PFC7515-B14-10 </td></tr><tr><td> Navigateur BioWorks </td><td> Thermo Scientific </td><td></td></tr></tbody></table>

quantitative analysis of chromatin proteomes in disease

Dans le noyau résident les protéomes dont les fonctions sont le plus intimement lié à la régulation des gènes. Adulte noyaux des cardiomyocytes de mammifères sont uniques en raison du pourcentage élevé de cellules binucléées, 1 l&#39;état hétérochromatique principalement de l&#39;ADN, et la nature non-division du cardiomyocyte qui rend noyaux adulte dans un état ​​permanent d&#39;interphase. 2 Régulation transcriptionnelle au cours du développement et maladie ont été bien étudiés dans cet organe, 3-5, mais ce qui reste relativement inexploré est le rôle joué par les protéines nucléaires responsables de l&#39;empaquetage de l&#39;ADN et de l&#39;expression, et comment ces protéines contrôlent des changements dans les programmes transcriptionnels qui se produisent au cours de la maladie. 6 Dans les pays développés monde, les maladies cardiovasculaires sont la première cause de mortalité pour les hommes et les femmes 7. Regard sur la façon dont les protéines nucléaires coopèrent pour réguler la progression de cette maladie est essentielle pour faire avancer le traitement actuel options. La spectrométrie de masse est l&#39;outil idéal pour répondre à ces questions car elle permet une annotation non biaisée de la quantification nucléaire protéome et relative de la façon dont l&#39;abondance de ces protéines varie avec la maladie. Bien qu&#39;il y ait eu plusieurs études protéomiques pour des complexes de protéines de mammifères nucléaires, 8-13 jusqu&#39;à récemment 14, on a eu qu&#39;une seule étude portant sur ​​le protéome cardiaque nucléaire, et considéré comme le noyau entier, plutôt que d&#39;explorer le protéome au niveau des compartiments nucléaires sous 15. En grande partie, ce manque de travail est due à la difficulté d&#39;isoler les noyaux cardiaque. Noyaux cardiaque ultérieure dans un semi-rigide et dense actine-myosine l&#39;appareil auquel ils sont reliés par plusieurs extensions du réticulum endoplasmique, dans la mesure où la contraction des myocytes modifie leur forme générale. 16 En outre, les cardiomyocytes sont 40% en volume 17 mitochondries qui necessitàTES enrichissement du noyau mis à part les autres organites. Ici, nous décrivons un protocole d&#39;enrichissement nucléaire cardiaque et un fractionnement supplémentaire en compartiments biologiquement pertinentes. En outre, nous avons détail les méthodes pour le label sans dissection spectrométrie de masse quantitative de ces fractions des techniques qui se prêtent à l&#39;expérimentation in vivo dans divers modèles animaux et des systèmes d&#39;organes où marquage métabolique n&#39;est pas réalisable.

La figure 4 met en évidence l&#39;utilité de cette forme de quantification relative. Montré dans le panneau de gauche sont différents pics peptidiques monoisotopiques (recouvert de différentes souris), qui ont été désignés comme appartenant à la HMGB1 protéines (identifiés par une recherche de base de données). Chaque pic, essentiellement un chromatographe ionique extrait pour le peptide donné, provient d&#39;une autre souris. Les groupes représentent les trois différents états physiolo...

Watch this Scientific Journal Video about Quantitative Analysis of Chromatin Proteomes in Disease at JoVE.com

Quantitative Analysis of Chromatin Proteomes in Disease

Les progrès de la spectrométrie de masse ont permis l&#39;analyse à haut débit de l&#39;expression protéique et la modification dans une multitude de tissus. Combiné avec fractionnement subcellulaire et modèles de maladies, spectrométrie de masse quantitative et de la bioinformatique peut révéler de nouvelles propriétés dans les systèmes biologiques. Le procédé décrit ici analyse des protéines associées à la chromatine dans le cadre d&#39;une maladie cardiaque et est facilement applicable à d&#39;autres<em&gt; In vivo</em&gt; Modèles de maladies humaines.

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Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

3D quantitative<em&gt; In Silico</em&gt; Modélisation (q3DISM) de Cerebral bêta-amyloïde phagocytose dans les modèles rongeurs de la maladie d&#39;Alzheimer

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

Application de l’analyse de causalité de Granger de la connexion fonctionnelle dirigée dans la maladie d’Alzheimer et des troubles cognitifs légers

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in MeniÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¨re's Disease Ears and Normal Hearing Ears

In Vivo Analyse morphométrique des humains nerfs crâniens à l’aide de l’imagerie par résonance magnétique oreilles maladie de Menière et des oreilles de l’audition normale

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRIÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚ÂApplication in Premanifest Huntington's Disease

Ensemble-cerveau Segmentation et analyse des points de changement d’IRM anatomique cérébrale — Application dans Premanifest la maladie de Huntington

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-aminopropionitrile-induced Aortic Aneurysm and Dissection

Analyse quantitative de Micro-CT de Aortopathy dans un modèle murin d’anévrisme de l’aorte induite par la β-aminopropionitrile et Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...