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 er støttet med tilskudd fra National Heart, Lung and Blood Institute of NIH og Laubisch Endowment ved UCLA. EM er mottaker av Jennifer S. Buchwald Graduate Fellowship i fysiologi ved UCLA, HC er mottaker av en American Heart Association Pre-doc, MP er mottaker av en NIH Ruth Kirschstein post-doc, og SF er mottaker av en NIH K99 Award.

Den eksperimentelle arbeidsflyt inneholder sju store trinn (figur 1). For noe arbeid som involverer prøver som skal kjøres på massespektrometer, bør eksperimentator bære en frakk, hansker og hårnett og ta vare å unngå forurensning fra støv og personlig Shedding av keratin. 1. Hjerte Homogenisering og Nuclear Isolation Muse hjerter er homogenisert og en intakt kjerner pellet er isolert (figur 2). <ol><li> Ofre voksen mu...

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To viktigste metodene for kjernefysisk isolasjon er gjennomgått tidligere: 27 en er Behrens teknikken homogenisere lyofilisert vev i en ikke-vandig løsningsmiddel og andre, er en modifikasjon som vi bruke her, av homogenisering vev i en vandig sukrose / saltoppløsning fulgt av differensial eller tetthet-gradient sentrifugering. Subfraksjonering av kjernene ved syreekstraksjon på vevsprøver er et viktig verktøy for å studere kromatin som har vært brukt siden 1960, 28</...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td></tr><tr><td> Dulbeco Forandringer Eagle Medium </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> Protease pellet </td><td> Roche </td><td> 04 693 159 001 </td></tr><tr><td> 100 mikrometer sil </td><td> BD Falcon </td><td> 352360 </td></tr><tr><td> ULTRACUT ultramicrotome </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> Histone H2A antistoff </td><td> Santa Cruz </td><td> sc-8648 </td></tr><tr><td> Nucleoporin p62 antistoff </td><td> BD Biosciences </td><td> 610498 </td></tr><tr><td> Adenin Nucleotidevannet transporter antistoff </td><td> Santa Cruz </td><td> sc-9299 </td></tr><tr><td> BIP antistoff </td><td> Santa Cruz </td><td> sc-1050 </td></tr><tr><td> Tubulin antistoff </td><td> Sigma </td><td> T1568 </td></tr><tr><td> Histone H3 antistoff </td><td> Abcam </td><td> ab1791 </td></tr><tr><td> Fibrillarin antistoff </td><td> Cellesignalisering </td><td> C12C3 </td></tr><tr><td> SNRP70 antistoff </td><td> Abcam </td><td> ab51266 </td></tr><tr><td> E2F-1 antistoff </td><td> Thermo Fisher </td><td> MS-879 </td></tr><tr><td> Retinoblastom antistoff </td><td> BD Biosciences </td><td> 554136 </td></tr><tr><td> Hypoksi induserbar faktor-1 antistoff </td><td> Novus Biologicals </td><td> NB100-469 </td></tr><tr><td> BCA protein assay </td><td> Thermo Scientific </td><td> 23227 </td></tr><tr><td> Reversfasekolonne </td><td> Ny Objektiv </td><td> PFC7515-B14-10 </td></tr><tr><td> BioWorks Browser </td><td> Thermo Scientific </td><td></td></tr></tbody></table>

quantitative analysis of chromatin proteomes in disease

I kjernen bor de proteomes hvis funksjoner er mest nært knyttet genregulering. Voksen pattedyr cardiomyocyte kjerner er unik på grunn av den høye prosentandel av binucleated celler, 1 har den overveiende heterochromatic tilstanden av DNA, og den ikke-delende natur cardiomyocyte som gjengir voksen atomkjerner i en permanent tilstand av interfase. 2 transkripsjonsregulering under utvikling og sykdom har blitt godt undersøkt i dette organet, 3-5, men det er fortsatt relativt uutforsket er den rollen de kjernefysiske proteiner ansvarlig for DNA emballasje og uttrykk, og hvordan disse proteinene styrer endringene i transcriptional programmer som oppstår under sykdom. 6 I den utviklede verden, er hjertesykdom nummer én årsak til dødelighet for både menn og kvinner. 7 innsikt i hvordan kjernefysiske proteiner samarbeide for å regulere utviklingen av denne sykdommen er avgjørende for å fremme den aktuelle behandlingen options. Massespektrometri er det ideelle verktøy for å håndtere disse spørsmålene som gjør det mulig for en objektiv annotering av den kjernefysiske proteomet og relativ kvantifisering for hvordan overflod av disse proteinene endringer med sykdom. Mens det har vært flere proteomikk studier for pattedyr kjernefysiske protein komplekser, 8-13 inntil nylig 14 har det vært bare en studie som undersøker hjertets kjernefysiske proteomet, og det anses hele kjernen, snarere enn å utforske proteomet på nivået av kjernefysiske sub avdelinger . 15 I stor grad er dette mangel på arbeid på grunn av vanskeligheter med å isolere hjerte kjerner. Cardiac atomkjerner forekommer innenfor en stiv og tett aktin-myosin apparat som de er forbundet via flere forlengelser fra det endoplasmatiske retikulum, i den grad at myocyte sammentrekning endrer deres samlede form. 16 Dessuten cardiomyocytes er 40% mitokondrier volum 17 som Necessitates anrikning av kjernen bortsett fra de andre organeller. Her beskriver vi en protokoll for hjerte kjernefysisk berikelse og videre fraksjonering i biologisk relevante avdelinger. Videre har vi detaljerte metoder for label-fri kvantitativ massespektrometrisk disseksjon av disse fraksjoner-teknikker mottagelig for in vivo eksperimenter i ulike dyremodeller og organsystemer der metabolske merking er ikke gjennomførbart.

Figur 4 viser nytten av denne formen for relativ kvantifisering. Som vises i venstre panel er de enkelte monoisotopic peptid toppene (kledde fra ulike mus), som har blitt utpekt som tilhører protein HMGB1 (identifisert via databasen søk). Hver topp, i hovedsak en ekstrahert ion kromatograf for den gitte peptid, kommer fra en annen mus. Gruppene representerer tre ulike fysiologiske tilstander: basal, kardial hypertrofi og hjertesvikt, med tre biologiske replikater for hver gruppe. Ved å integrere area...

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

Quantitative Analysis of Chromatin Proteomes in Disease

Fremskritt innen massespektrometri har tillatt høy gjennomstrømning analyse av protein uttrykk og modifikasjon på en rekke vev. Kombinert med subcellulære fraksjonering og sykdom modeller, kvantitative massespektrometri og bioinformatikk kan avsløre nye egenskaper i biologiske systemer. Metoden er beskrevet her analyserer kromatin-assosierte proteiner i innstillingen av hjertesykdom og er lett anvendelig til andre<em&gt; In vivo</em&gt; Modeller av sykdom hos mennesker.

Kvantitativ analyse av Chromatin Proteomes i sykdom

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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Anvendelsen av Granger kausalitet analyse av regissert funksjonelle tilkoblingen i Alzheimers sykdom og Mild kognitiv svekkelse

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

I Vivo Morphometric analyse av menneskelig Hjernenerve bruker magnetisk resonans Imaging i Menières sykdom ører og Normal hørsel ører

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

Hele-hjerne segmentering og viser analyse av anatomiske hjernen MRI-programmet i Premanifest Huntingtons sykdom

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

Kvantitativ mikro-CT analyse av Aortopathy i en musemodell av β-aminopropionitrile-indusert aortaaneurisme og disseksjon

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