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

Den Vondriska Lab stöds av anslag från National Heart, Lung and Blood Institute av NIH och Laubisch Endowment vid UCLA. EM är mottagare av Jennifer S. Buchwald Graduate Fellowship i fysiologi vid UCLA, HC är mottagare av ett American Heart Association doktorander Fellowship, MP är mottagare av en NIH Ruth Kirschstein postdoktorala stipendiet, och SF är mottagare av en NIH K99 Award.

Den experimentella arbetsflödet innehåller sju stora steg (Figur 1). För varje arbete med prover som ska köras på masspektrometern bör försöksledaren bära labbrock, handskar och hårnät och vara noga med att undvika kontamination från damm och personlig utsöndring av keratin. 1. Hjärta Homogenisering och Nuclear Isolering Mus hjärtan homogeniseras och en intakt kärnor pelleten isoleras (figur 2). <ol><li> Offra v...

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Två huvudsakliga metoder för kärn isolering har granskats tidigare: 27 en är Behrens teknik homogenisering lyofiliserat vävnad i ett icke-vattenhaltigt lösningsmedel och andra, en modifiering som vi använder här, homogenisering vävnad i en vattenbaserad sackaros / saltlösning följt genom differentiell eller densitet-centrifugering. Subfraktionering av kärnorna genom syraextraktion på vävnadsprov är ett viktigt verktyg för att studera kromatin som har använts sedan...

<table border="1"><tbody><tr><td> Namnet av reagenset </td><td> Företag </td><td> Katalognummer </td></tr><tr><td> Dulbeco Modified Eagle Medium </td><td> Invitrogen </td><td> 11.965 </td></tr><tr><td> Proteas pellets </td><td> Roche </td><td> 04 693 159 001 </td></tr><tr><td> 100 pm sil </td><td> BD Falcon </td><td> 352.360 </td></tr><tr><td> Ultracut ultramikrotom </td><td> Reichert </td><td></td></tr><tr><td> 100CX Transmission Electron Mikroskop </td><td> JEOL USA, Inc. </td><td></td></tr><tr><td> Oriole </td><td> BioRad </td><td> 161-0496 </td></tr><tr><td> Histon H2A antikropp </td><td> Santa Cruz </td><td> sc-8648 </td></tr><tr><td> Nucleoporin P62-antikropp </td><td> BD Biosciences </td><td> 610.498 </td></tr><tr><td> Adenin Nucleotidvatten transportör antikropp </td><td> Santa Cruz </td><td> sc-9299 </td></tr><tr><td> BiP antikropp </td><td> Santa Cruz </td><td> SC-1050 </td></tr><tr><td> Tubulin antikropp </td><td> Sigma </td><td> T1568 </td></tr><tr><td> Histon H3 antikropp </td><td> Abcam </td><td> ab1791 </td></tr><tr><td> Fibrillarin antikropp </td><td> Cellsignalering </td><td> C12C3 </td></tr><tr><td> SNRP70 antikropp </td><td> Abcam </td><td> ab51266 </td></tr><tr><td> E2F-1-antikropp </td><td> Thermo Fisher </td><td> MS-879 </td></tr><tr><td> Retinoblastom antikropp </td><td> BD Biosciences </td><td> 554.136 </td></tr><tr><td> Hypoxi inducerbara faktor-1-antikropp </td><td> Novus Biologicals </td><td> NB100-469 </td></tr><tr><td> BCA-proteinanalys </td><td> Thermo Scientific </td><td> 23.227 </td></tr><tr><td> Omvänd fas-kolonn </td><td> Nya Objektiva </td><td> PFC7515-B14-10 </td></tr><tr><td> BioWorks webbläsare </td><td> Thermo Scientific </td><td></td></tr></tbody></table>

quantitative analysis of chromatin proteomes in disease

I kärnan bor på proteom vars funktioner är mest intimt förknippade med genreglering. Vuxen däggdjur cardiomyocyte kärnor är unika på grund av den höga andelen binucleated celler, 1 den övervägande heterochromatic tillstånd DNA, och icke delande natur cardiomyocyte som gör vuxna kärnor i ett permanent tillstånd av interfas. 2 Transkriptionsreglering under utveckling och sjukdom har studerats i detta organ, 3-5, men det som återstår relativt outforskat är den roll som spelas av nukleära proteiner som är ansvariga för DNA-förpackning och uttryck och hur dessa proteiner kontrollerar förändringar i transkriptionella program som inträffar under sjukdom. I utvecklade 6 världen är hjärtsjukdom den vanligaste dödsorsaken för både män och kvinnor. 7 insikt om hur kärnproteiner samverkar för att reglera utvecklingen av denna sjukdom är avgörande för att föra den aktuella behandlingen o= Val. Masspektrometri är det perfekta verktyget för ta itu med dessa frågor, eftersom det möjliggör en opartisk anteckning av kärn proteomet och relativa kvantifiering för hur överflöd av dessa proteiner förändras med sjukdom. Även om det har gjorts flera proteomik studier för däggdjur nukleära proteinkomplex, 8-13 tills nyligen 14 har det varit bara en studie för att undersöka hjärtats nukleära proteom, och det ansåg hela kärnan, snarare än att utforska proteomet i nivå med nukleära sub fack . 15 Till stor del är denna brist på arbete på grund av svårigheten att isolera hjärt kärnor. Hjärt kärnor sker inom en styv och tät aktin-myosin apparat som de är anslutna via flera förlängningar från det endoplasmatiska retiklet, i den utsträckning som myocyt kontraktion förändrar deras totala form. 16 Dessutom, kardiomyocyter är 40% mitokondrier volym 17 vilken necessitaTES anrikning av kärnan bortsett från de andra organeller. Här beskriver vi ett protokoll för hjärt anrika uran och ytterligare fraktionering i biologiskt relevanta delar. Dessutom har vi detalj metoder för märkning utan kvantitativa masspektrometrisk dissektion av dessa fraktioner-tekniker som lämpar sig för in vivo-experiment i olika djurmodeller och organsystem där metabolisk märkning inte är möjlig.

Figur 4 belyser användbarheten av denna form av relativ kvantifiering. Visas i den vänstra panelen är de individuella monoisotopisk peptid toppar (överlagras från olika möss), som har utsetts som tillhör protein HMGB1 (identifierad genom databassökning). Varje topp, väsentligen en extraherade jonkromatograf för den givna peptiden, kommer från en annan mus. Grupperna representerar tre olika fysiologiska tillstånd: basal, hjärthypertrofi och hjärtsvikt, med tre biologiska replikat för varje...

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

Quantitative Analysis of Chromatin Proteomes in Disease

Framsteg inom masspektrometri har tillåtit hög genomströmning analys av proteinuttryck och modifiering i en mängd vävnader. Kombinerat med subcellulär fraktionering och modeller sjukdom, kvantitativ masspektrometri och bioinformatik kan avslöja nya egenskaper i biologiska system. Det häri beskrivna förfarandet analyserar kromatin-associerade proteiner i inställningen av hjärtsjukdomar och är lätt tillämpas på andra<em&gt; In vivo</em&gt; Modeller av mänsklig sjukdom.

Kvantitativ analys av kromatin proteom vid sjukdom

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

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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

Tillämpningen av Granger kausalitet analys av riktad funktionella anslutningen i Alzheimers sjukdom och kognitiv svikt

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 Morfometriska analys av mänskliga kranialnerver med magnetkamera i Menières sjukdom öron och Normal hörsel öron

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

Hela-hjärnan segmentering och ändra punkt analys av anatomiska hjärnan MRI — tillämpning i Premanifest Huntingtons sjukdom

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

Kvantitativa Micro-CT analys av Aortopathy i en musmodell av β-aminopropionitrile-inducerad aorta Aneurysm och dissektion

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