Name: a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation
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Description: Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

Vi takker Karyn Esser, Nobuya Koike og Noheon Park for nyttige råd. Dette arbejde blev delvist støttet af NIH / NIGMS (R01GM114424) til S.-HY, og Robert A. Welch Foundation (AU-1731) og NIH / NIA (R01AG045828) til ZC

Dyrpleje blev udført under retningslinierne for Institutional Animal Care and Use Committee (IACUC), og procedurerne blev udført i henhold til en animalsk protokol godkendt af University of Texas Health Science Center i Houston. 1. Nuclei-isolation fra tværbundet skeletmuskel <ol><li> Skeletmuskulatur af vægt og bagvæg, isoleret fra ca. 20 ugers gamle C57BL / 6 hanmus, i iskold fosfatbuffet saltvand (PBS) som beskrevet detaljeret tidligere 22 . BEMÆRK: ...

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Her beskriver vi en robust metode, hvor tværbundet skeletmuskelvæv blev brugt til at isolere kerne af høj kvalitet. Sekventiel filtrering blev udført for effektivt at adskille kerner fra affald, og ultralydsakustisk energi fra skålformet transducer skåret kromatinet til ChIP-analyse. Resultaterne viste cirkadisk tidsspecifik binding af BMAL1 til målpromotorer. ChIP kan anvendes til at indfange realtidsproteinbelægning på genomisk DNA, når tværbindingen finder sted. For at udnytte ...

Skeletmuskel spiller vigtige roller i fysiologi og adfærd. Den multikernerede muskelfiber består af myofibriller, hvor actin og myosin danner funktionelle enheder kaldet sarcomerer for at danne kontraktilkraft. Skeletmuskler er også det største stofskifteorgan i kroppen, der tegner sig for&gt; 80% postprandial glukoseindtagelse og regulerer insulinrespons og metabolisk homeostase 1 , 2 . Muskelfysiologi og stofskifte er tæt reguleret af det cirkadiske ur, en iboende biologisk timer 3 , 4 , 5 , 6 . For eksempel resulterede den skeletmuskelspecifikke deletion af Bmal1 , en af ​​de kernekredittiske urkomponenter , insulinresistens og nedsat glukoseoptagelse i skeletmuskel, og dyrene viste sig at udvikle type 2-diabetes 7 . jegN-tillæg bliver skeletmuskel også i stigende grad værdsat som et hormon 8 , der udskiller myokiner for at regulere systemisk metabolisme og fysiologi. Mekanistiske undersøgelser er nødvendige for fuldt ud at forstå disse regulatoriske funktioner i skeletmuskel. ChIP er en kraftfuld tilgang til afgrænsning af promotor rekruttering af DNA-bindende proteiner. ChIP blev oprindeligt udviklet til at identificere nukleosomorganisering på chromatin DNA 9 , 10 . En række fremgangsmåder er siden blevet rapporteret til tværbindingsproteiner og chromatin-DNA ved anvendelse af formaldehyd, dimethylsulfat eller ultraviolet bestråling (UV) 11 , 12 . Formaldehyd-tværbinding er den mest almindeligt anvendte, konserverende kromatinstruktur og DNA-proteininteraktioner 9 , 13 , 14 . Tværbundet kromatografiIn er shredded ved sonication og immunpræcipiteret med antistof mod det specifikke DNA-bindende protein af interesse 15 , 16 . I de senere år er ChIP-sekventering (ChIP-seq), en metode, der kombinerer ChIP med NGS, udviklet for at forhøre genomfattende transkriptionsfaktorbinding 17 og i nogle tilfælde at overvåge dynamiske ændringer over et tidsforløb 18 , 19 , 20 . For eksempel har circadian-ChIP-seq-studier afsløret en stærkt orkestreret sekvens af genomisk binding af kredsløbskomponenter og histonmarkører, som dirigerer temporært præcis genekspression gennem hele den 24-timers cirkadiske cyklus 18 . De fleste tilgængelige ChIP-protokoller er designet til blødt væv ( fx lever, hjerne osv. ), Og meget få er blevet offentliggjort til hårdt væv inklusive skeleTal muskler. Det er teknisk udfordrende at homogenisere fiberrig skeletmuskel og isolere højkvalitets kerne 21 , især til ChIP-eksperimenter, som kræver tværbinding. I en nylig muskel-ChIP-undersøgelse 22 blev satellitceller adskilt fra myofibere, og kerner blev fremstillet ud fra begge celletyper gennem en forlænget procedure, der involverer vævsfordøjelse. Hele processen tog cirka tre timer at afslutte før formaldehyd-tværbinding blev udført på isolerede kerner. Selvom denne procedure undgik krydsbinding af muskelfibre, hvilket gør muskelvæv endnu mere ildfast til effektiv homogenisering og var i stand til at producere kerne af høj kvalitet, medfører den betydelige tidsforsinkelse fra vævsopsamling til kerneforbindelser risikoen for ændret DNA -proteininteraktion. I modsætning hertil udførte de fleste undersøgelser tværbinding umiddelbart efter forsøgsbehandling eller vævsopsamling for at bevare real-time DNA-proteiN bindende 12 . En anden ulempe ved kerneisolation før tværbinding er, at den udelukker tidsfølsomme anvendelser såsom cirkadisk prøveopsamling, som typisk forekommer med intervaller på 3 - 4 timer. Uden krydsbinding af kernerne skal isolationen fortsætte umiddelbart efter dissektion, mens tværbundne prøver kan behandles sammen, efter at hele tidskursus er afsluttet, hvilket sikrer større eksperimentel konsistens. Andre protokoller for kerneisolation fra ikke-tværbundet skeletmuskel er også blevet rapporteret. To undersøgelser beskrev brugen af ​​gradient ultracentrifugering til adskillelse af kerner fra resterende myofibriller og celleaffald 23 , 24 . Selvom sukrose eller kolloidal gradient ultracentrifugering er effektiv med ikke-tværbundne muskelvæv, viste vores eksperimenter, at efter krydsbinding mislykkedes gradient-ultracentrifugering til at adskille kerner fra celle dEbris på graden. Vi udviklede derfor en procedure til isolering af kerne af høj kvalitet ved anvendelse af tværbundne muskelvæv i muskelskelet. I stedet for gradient ultracentrifugering udtænkte vi en seriefiltreringsmetode til effektivt at adskille kerner fra affald. Efter ultralydning blev chromatinprøverne succesfuldt anvendt til ChIP-undersøgelser, der viste et cirkadisk mønster af BMAL1-proteinbinding til målpromotorer. Vores metode kan bredt anvendes til forskellige mekaniske studier af muskelvæv.

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			<td height="20" style="height:20px;width:216px;">Materials</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Formaldehyde solution</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">F8775&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glycine</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">BP381-5</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Trypan Blue solution (0.4%)</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
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			<td height="20" style="height:20px;width:216px;">RNase A</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">10109142001</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Protease K</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">3115887001</td>
			<td></td>
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		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Chicken Anti-BMAL1 antibody</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:288px;">Generated in chicken (Cocalico Biologicals) against antigen aa 318-579, and IgY was affinity purified using the same antigen.&#160;</td>
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			<td height="20" style="height:20px;width:216px;">Chicken IgY Precipitating Resin</td>
			<td style="width:143px;">GenScript</td>
			<td style="width:119px;">L00405</td>
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			<td height="20" style="height:20px;width:216px;">Equipment</td>
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			<td height="38" style="height:38px;width:216px;">KINEMATICA&#160;Polytron PT2100 Benchtop Homogenizer</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-451-178</td>
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			<td height="20" style="height:20px;width:216px;">15mL Dounce tissue grinder</td>
			<td style="width:143px;">Whearton</td>
			<td style="width:119px;">357544</td>
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			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 100&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-19</td>
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			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 70&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-1</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 40&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 30 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50030-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 20 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50020-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 10 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50010-03</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Covaris S2 Focused-ultrasonicator</td>
			<td style="width:143px;">Covaris</td>
			<td style="width:119px;">Model S2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Labquake&#160;</td>
			<td>Thermo Scientific</td>
			<td>C415110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD Microscope Camera</td>
			<td>&#160;Leica Microsystems</td>
			<td>DFC3000 G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Reagent Kit</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DNA extraction kit</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:119px;">K0691</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Buffers</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation

Kromatinimmunudfældning (ChIP) er en kraftfuld metode til bestemmelse af proteinbinding til chromatin-DNA. Fiber-rige skeletmuskler har imidlertid været en udfordring for ChIP på grund af tekniske vanskeligheder i isolation af højkvalitetskerner med minimal forurening af myofibriller. Tidligere protokoller har forsøgt at rense kerner inden krydsbinding, hvilket medfører risikoen for ændret DNA-proteininteraktion under den forlængede kerneforberedelsesproces. I den nuværende protokol tværbindede vi først skeletmuskelvævet opsamlet fra mus, og vævene blev hakket og sonikeret. Da vi fandt ud af, at ultracentrifugering ikke var i stand til at adskille kerner fra myofibriller ved anvendelse af tværbundet muskelvæv, udtænkte vi en sekventiel filtreringsprocedure for at opnå højkvalitets kerner uden væsentlig myofibrilforurening. Vi fremstillede efterfølgende chromatin ved anvendelse af en ultralydator, og ChIP-assays med anti-BMAL1-antistof afslørede robust cirkadisk bindingMønster af BMAL1 for at målrette genpromotorer. Denne filtreringsprotokol udgør en let anvendelig metode til isolering af kerne af høj kvalitet fra tværbundet skeletmuskulaturvæv, hvilket muliggør konsekvent prøvebehandling til cirkadian og andre tidsfølsomme undersøgelser. I kombination med næste generations sekventering (NGS) kan vores metode implementeres til forskellige mekaniske og genomiske undersøgelser med fokus på skeletmuskelfunktion.

Her gennemførte vi formaldehyd-tværbinding umiddelbart efter vævsopsamling for at bevare real-time DNA-protein interaktion. Vi fandt imidlertid, at saccharose eller kolloidal gradient, der almindeligvis anvendes til nuclei-isolering 23 , 24 , ikke var effektiv til at adskille kerner fra myofibriller (data ikke vist). Årsagen kan være, at tværbinding gav samme tyngdekraft for kerner og myofibriller. Derfor udviklede vi en se...

Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Vi præsenterer en filtreringsbaseret protokol til isolering af kerne af høj kvalitet fra tværbundet museskeletmuskulatur, hvor vi fjernede behovet for ultracentrifugering, hvilket gør det let anvendeligt. Vi viser, at kromatin fremstillet ud fra kernerne er egnet til chromatinimmunpræcipitation og sandsynligvis chromatinimmunpræcipitationssekventeringsundersøgelser.

En filtreringsbaseret metode til fremstilling af højkvalitetsnuclei fra tværbundet skeletsmuskel til chromatinimmunpræcipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Research

JoVE Journal

Biochemistry

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

En Facile og effektiv tilgang til fremkaldelse af reversibel disulfid Cross-forbundne Miceller

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

En enkel fraktioneret ekstraktionsmetode til den omfattende analyse af metabolitter, lipider og proteiner fra en enkelt prøve

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Forbedret protokol for kromatin Immunoprecipitation fra mus skeletmuskulatur

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

Aktiveret tvær kædet Agopstået for den hurtige udvikling af affinitets kromatografi harpiks-antistof opsamling som et casestudie

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Kvantificering af protein interaktions netværk dynamik ved hjælp af Multiplexed Co-Immunopræcipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Preparation of High-Quality Fermented Fish Product

Tilberedning af fermenteret fiskeprodukt af høj kvalitet

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

Opnåelse af udskriftsdata af høj kvalitet fra kornfrø ved en modificeret metode til genekspressionsprofilering

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Forberedelse af lameller fra glasagtige biologiske prøver ved hjælp af et dobbeltstrålescanningselektronmikroskop til kryo-elektrontomografi

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Måling af proteinimportkapacitet af skeletmuskulatur mitokondrier

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

Kvantificering af subcellulær glykogenfordeling i skeletmuskelfibre ved hjælp af transmissionselektronmikroskopi

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...