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 af tilskud fra National Heart, Lung og Blood Institute of NIH og Laubisch Endowment på UCLA. EM er modtager af Jennifer S. Buchwald Graduate Fellowship i fysiologi ved UCLA, HC er modtageren af ​​en American Heart Association Pre-ph.d Fellowship, MP er modtageren af ​​en NIH Ruth Kirschstein Post-ph.d. stipendium, og SF er modtageren af en NIH K99 Award.

Den eksperimentelle workflow indeholder syv store trin (figur 1). For ethvert arbejde, der involverer prøver, der vil blive kørt på massespektrometer, bør eksperimentatoren bære en laboratoriekittel, handsker og hårnet og sørge for at undgå forurening fra støv og personlig udgydelse af keratin. 1. Heart Homogenisering og nuklear Isolation Mus hjerter homogeniseres og et intakt kerner pelleten isoleret (fig. 2). <ol><li...

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To primære fremgangsmåder til nuklear isolation er gennemgået tidligere: 27 ene er den Behrens teknik homogenisering lyofiliseret væv i et ikke-vandigt opløsningsmiddel, og den anden, en modifikation, som vi bruger her, for at homogenisere vævet i en vandig saccharose / saltopløsning efterfulgt ved differentiel eller densitet-gradientcentrifugering. Subfraktionering af kernerne ved syreekstraktion på vævsprøver er et vigtigt redskab til undersøgelse af kromatin, som har...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Dulbeco Modified Eagle Medium </td><td> Invitrogen </td><td> 11.965 </td></tr><tr><td> Protease pellet </td><td> Roche </td><td> 04 693 159 001 </td></tr><tr><td> 100 um si </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 Microscope </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 antistof </td><td> Santa Cruz </td><td> sc-8648 </td></tr><tr><td> Nucleoporin p62 antistof </td><td> BD Biosciences </td><td> 610.498 </td></tr><tr><td> Adenin Nucleotidevand transporter antistof </td><td> Santa Cruz </td><td> sc-9299 </td></tr><tr><td> BiP antistof </td><td> Santa Cruz </td><td> sc-1050 </td></tr><tr><td> Tubulin antistof </td><td> Sigma </td><td> T1568 </td></tr><tr><td> Histon H3-antistof </td><td> Abcam </td><td> ab1791 </td></tr><tr><td> Fibrillarin antistof </td><td> Cellesignalering </td><td> C12C3 </td></tr><tr><td> SNRP70 antistof </td><td> Abcam </td><td> ab51266 </td></tr><tr><td> E2F-1-antistof </td><td> Thermo Fisher </td><td> MS-879 </td></tr><tr><td> Retinoblastom antistof </td><td> BD Biosciences </td><td> 554.136 </td></tr><tr><td> Hypoksi inducible factor-1-antistof </td><td> Novus Biologicals </td><td> NB100-469 </td></tr><tr><td> BCA-proteinassay </td><td> Thermo Scientific </td><td> 23.227 </td></tr><tr><td> Omvendt fase-søjle </td><td> Ny Objective </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 kernen bor de proteomes hvis funktioner er mest tæt knyttet til genregulering. Voksent pattedyr cardiomyocyte kerner er unik på grund af den høje procentdel af binucleated celler, 1 den overvejende heterochromatic tilstand af DNA&#39;et, og den ikke-delende art cardiomyocyte som gør voksne kerner i en permanent tilstand af interfase. 2 Transkriptionel regulering under udvikling og sygdom er blevet godt undersøgt i dette organ, 3-5, men hvad forbliver relativt uudforsket, er den rolle, som de nukleare proteiner, der er ansvarlige for DNA emballage og udtryk, og hvordan disse proteiner styre ændringer i transkriptionelle programmer, der opstår under sygdom. 6 I den udviklede verden, hjertesygdom er den største årsag til dødelighed for både mænd og kvinder. 7 indsigt i, hvordan kerneproteiner samarbejder om at regulere udviklingen af denne sygdom er afgørende for at fremme den nuværende behandling oULIGHEDER. Massespektrometri er det ideelle redskab til at løse disse spørgsmål, da det giver mulighed for en fordomsfri annotation af det nukleare proteomanalyse og relativ kvantificering for hvordan overflod af disse proteiner ændringer med sygdom. Mens der har været flere proteom undersøgelser for mammale nukleare proteinkomplekser, 8-13 indtil for nylig 14 har der kun været én undersøgelse undersøger hjertets nukleare proteom, og den fandt hele kerne, snarere end at udforske proteomet på niveau med nukleare sub rum . 15. I stor del, dette arbejdsmangel skyldes vanskeligheden ved at isolere hjerte kerner. Cardiac kerner forekommer inden for et stift og tæt actin-myosin apparatur, hvortil de er forbundet via flere udvidelser fra det endoplasmatiske reticulum, i det omfang, myocyt sammentrækning ændrer deres samlede form. Seksten Derudover kardiomyocytter er 40% mitokondrier vol. 17, som necessitaTES berigelse af kernen bortset fra de andre organeller. Her beskriver vi en protokol for hjerte-nukleare berigelse og yderligere fraktionering i biologisk relevante rum. Endvidere har vi detalje metoder til label-fri kvantitativ massespektrometrisk dissektion af disse fraktioner-teknikker modtagelige for in vivo eksperimenter i forskellige dyremodeller og organsystemer hvor metabolisk mærkning ikke er mulig.

Figur 4 fremhæver nytten af denne form for relativ kvantificering. Vises i venstre panel er de enkelte monoisotopic peptidtoppe (overlejret fra forskellige mus), som er blevet udpeget som hørende til proteinet HMGB1 (identificeret via databasesøgning). Hver top, hovedsagelig en ekstraherede ion chromatograf for den givne peptid, kommer fra en anden mus. Grupperne repræsenterer tre forskellige fysiologiske tilstande: basal, hjertehypertrofi og hjertesvigt, med tre biologiske replikater for hver grupp...

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

Quantitative Analysis of Chromatin Proteomes in Disease

Fremskridt inden massespektrometri har tilladt high throughput analyse af proteinekspression og ændringen i en lang række væv. Kombineret med subcellulær fraktionering og sygdomsmodeller, kvantitativ massespektrometri og bioinformatik kan afsløre nye egenskaber i biologiske systemer. Fremgangsmåden beskrevet heri analyseres chromatin-associerede proteiner i tilfælde af hjertesygdomme og let anvendes til andre<em&gt; In vivo</em&gt; Modeller af sygdomme hos mennesker.

Kvantitativ analyse af kromatin Proteomes i Disease

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 af Granger kausalitet analyse af styret funktionelle forbindelsen i Alzheimers sygdom og Mild kognitiv svækkelse

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 Morfometrisk analyse af menneskers hjernenerver bruger magnetisk resonans i Menières sygdom ører og Normal hørelse 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 Change-point analyse af anatomiske hjerne MRI-program i premanifeste Huntington's chorea

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

Kvantitative mikro-CT-analyse af Aortopathy i en musemodel af β-aminopropionitrile-induceret aorta aneurisme og dissektion

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