Name: analysis of the c-kit ligand promoter using chromatin immunoprecipitation
Uploaded: 2017-06-27T15:00:00
Description: Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

Detta arbete stöddes av University of Texas MD Anderson Cancer Center, Houston, TX (startfonder, BB).

1. Framställning av lösningar <ol><li> Förbered följande lösningar färskt för varje experiment. <ol><li> För fixeringslösningen , förbered 37% formaldehyd och förvara den vid RT. Späd det i 1x fosfatbuffrad saltlösning (PBS) till en slutlig koncentration av 1,42%. VARNING: Formaldehyd klassificeras som irriterande, frätande, mutagena, teratogena och cancerframkallande. Förtär inte. Andas inte in gas / ånga / ånga / spray. Vid otillräcklig ventilation, använd...

<ol>
	<li>Brenowitz, M., Senear, D. F., Shea, M. A., Ackers, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+DNase+footprint+titration:+a+method+for+studying+protein-DNA+interactions.">Quantitative DNase footprint titration: a method for studying protein-DNA interactions.</a> Methods Enzymol. 130, 132-181 (1986).</li><li>Garner, M. M., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions:+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system.">A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.</a> Nucleic Acids Res. 9 (13), 3047-3060 (1981).</li><li>Gilmour, D. S., Lis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+protein-DNA+interactions+in+vivo:+distribution+of+RNA+polymerase+on+specific+bacterial+genes.">Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.</a> Proc Natl Acad Sci USA. 81 (14), 4275-4279 (1984).</li><li>Gilmour, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lis+J.T.+In+vivo+interactions+of+RNA+polymerase+II+with+genes+of+Drosophila+melanogaster.">Lis J.T. In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.</a> Mol Cell Biol. 5 (8), 2009-2018 (1985).</li><li>Mundade, R., Ozer, H. G., Wei, H., Prabhu, L., Lu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ChIP-seq+in+the+discovery+of+transcription+factor+binding+sites,+differential+gene+regulation+mechanism,+epigenetic+marks+and+beyond.">Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond.</a> Cell Cycle. 13 (18), 2847-2852 (2014).</li><li>Mukhopadhyay, A., Deplancke, B., Walhout, A. J., Tissenbaum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+(ChIP)+coupled+to+detection+by+quantitative+real-time+PCR+to+study+transcription+factor+binding+to+DNA+in+Caenorhabditis+elegans.">Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans.</a> Nat Protoc. 3 (4), 698-709 (2008).</li><li>Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+chromatin+immunoprecipitation+to+clone+novel+E2F+target+promoters.">Use of chromatin immunoprecipitation to clone novel E2F target promoters.</a> Mol Cell Biol. 21 (20), 6820-6832 (2001).</li><li>Weinmann, A. S., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+unknown+target+genes+of+human+transcription+factors+using+chromatin+immunoprecipitation.">Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation.</a> Methods. 26 (1), 37-47 (2002).</li><li>Weinmann, A. S., Yan, P. S., Oberley, M. J., Huang, T. H., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+human+transcription+factor+targets+by+coupling+chromatin+immunoprecipitation+and+CpG+island+microarray+analysis.">Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis.</a> GenesDev. 16 (2), 235-244 (2002).</li><li>Massague, J., Seoane, J., Wotton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad+transcription+factors.">Smad transcription factors.</a> Genes Dev. 19 (23), 2783-2810 (2005).</li><li>Rojas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Positive+TGF-beta/c-KIT+Feedback+Loop+Drives+Tumor+Progression+in+Advanced+Primary+Liver+Cancer.">A Positive TGF-beta/c-KIT Feedback Loop Drives Tumor Progression in Advanced Primary Liver Cancer.</a> Neoplasia. 18 (6), 371-386 (2016).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nucleic+acids+by+extraction+with+phenol:chloroform.">Purification of nucleic acids by extraction with phenol:chloroform.</a> CSH Protoc. 2006 (1), (2006).</li><li>Dennler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+of+Smad3+and+Smad4+to+critical+TGF+beta-inducible+elements+in+the+promoter+of+human+plasminogen+activator+inhibitor-type+1+gene.">Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.</a> EMBO J. 17 (11), 3091-3100 (1998).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+Smad+MH1+domain+bound+to+DNA:+insights+on+DNA+binding+in+TGF-beta+signaling.">Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling.</a> Cell. 94 (5), 585-594 (1998).</li><li>Seidel, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spacing+of+palindromic+half+sites+as+a+determinant+of+selective+STAT+(signal+transducers+and+activators+of+transcription)+DNA+binding+and+transcriptional+activity.">Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity.</a> Proc Natl Acad Sci USA. 92 (7), 3041-3045 (1995).</li><li>Nelson, J. D., Denisenko, O., Bomsztyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+fast+chromatin+immunoprecipitation+(ChIP)+method.">Protocol for the fast chromatin immunoprecipitation (ChIP) method.</a> Nat Protoc. 1 (1), 179-185 (2006).</li><li>Tian, B., Yang, J., Brasier, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+cross-linking+for+analysis+of+protein-chromatin+interactions.">Two-step cross-linking for analysis of protein-chromatin interactions.</a> Methods Mol Biol. 809, 105-120 (2012).</li><li>O'Neill, L. P., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+native+chromatin:+NChIP.">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31 (1), 76-82 (2003).</li><li>Horz, W., Altenburger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+specific+cleavage+of+DNA+by+micrococcal+nuclease.">Sequence specific cleavage of DNA by micrococcal nuclease.</a> Nucleic Acids Res. 9 (12), 2643-2658 (1981).</li><li>Chung, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+micrococcal+nuclease+digestion+on+nucleosome+positioning+data.">The effect of micrococcal nuclease digestion on nucleosome positioning data.</a> PLoS One. 5 (12), 15754 (2010).</li></ol>

I denna rapport demonstrerar vi den TGF-β1-inducerade bindningen av SMAD2 till en SBE inom c-KIT ligandpromotorn och TGF-P1-inducerad bindning av STAT3 till dess igenkänningssekvens inom TGF-P1-ligandgenen. Vi demonstrerar cytokininducerad bindning av båda transkriptionsfaktorerna genom användning av kromatinimmunutfällning. Kromatinimmunutfällning är ett kraftfullt verktyg för att demonstrera direkt bindning av ett protein av intresse för DNA, för att karakterisera stimuli som ind...

I kärnan av eukaryoter interagerar DNA med histonproteiner och nonhistonproteiner och kondenseras till kromatin. I det fysiologiska, patofysiologiska och cellbiologiska sammanhanget kontrolleras cellfunktionerna rumligt och temporärt av kromatinkoordinerat genuttryck. DNA-protein-interaktioner har en väsentlig roll vid reglering av cellulära processer, såsom DNA-replikation, rekombination och reparation, såväl som proteinuttryck. Därför är analysen av DNA-protein-interaktioner ett oumbärligt verktyg vid utvärderingen av genuttryck och cellfunktion. Flera tekniker existerar för att bedöma DNA-protein-interaktioner in vitro , såsom elektrofiles (gel) Mobility Shift Assay (EMSA) och DNase I fotavtryck 1 , 2 . Emellertid analyserar dessa tekniker inte protein-DNA-interaktionen inom kromatin och cellulärt sammanhang. ChIP iEn teknik som fångar proteiner bundna till deras specifika DNA-bindningsställen och därmed underlättar identifieringen av DNA-protein-interaktioner inom kromatinkontexten. Tekniken var ursprungligen utvecklad av Gimour och Lis för bedömningen av RNA-polymeras II-bindning till specifika gener i Escherichia coli och Drosophila melanogaster 3 , 4 . Det görs genom fixering av DNA-proteinkomplexen, följt av utmatning av kromatin och skjuvning av DNA i ~ 200 baspar (bp) -fragment. Därefter isoleras det DNA-bundna proteinet av intresse genom immunutfällning. Efter reversering av DNA-proteintvärbindningen renas och analyseras DNA. Flera metoder kan användas för analys av proteinbindningsställena och beror på nukleinsyrasekvensen hos proteinbindningsstället inom målgenen 5 . I fall där DNA-sekvensen är känd, standardpolYmeras kedjereaktioner (PCR) kan appliceras, med användning av specifika primerpar som flankerar det kända bindningsstället. Kvantitativ realtid PCR (qRT-PCR) kan också användas 6 . I fall där sekvensen är okänd kan ChIP kombineras med DNA-mikroarrays (ChIP-on-chip), DNA-sekvensering (ChIP-seq) eller kloningsteknik 7 , 8 , 9 . TGF-P-vägen har kraftiga tumörundertryckande funktioner och är en nyckelväg i celldifferentiering. Det aktiveras genom bindning av TGF-P1-liganden till dess kognitiva receptorkomplex, vilket resulterar i serin-fosforylering av SMAD2 / 3-transkriptionsfaktorer. Efter deras förening med den gemensamma mediatorn, SMAD4, translaterar SMAD-komplexet till kärnan och binder till SBE inom promotorregionen hos målgenerna, där det reglerar gener som kontrollerar cellcykeln, apoptos och celldifferentieringenpå. Det transkriptionella svaret på TGF-p-stimulering är celltyp- och kontextspecifik 10 . Nyligen har vi beskrivit en positiv återkopplingsslinga mellan TGF-p och c-KIT-vägen 11 . I denna modell binder TGF-p1-aktiverad SMAD2 till c-KIT ligandpromotorn och inducerar dess uttryck och utsöndring. Därefter aktiverar c-KIT-liganden c-KIT-receptorn på ett auto-och para-kriniskt sätt. C-KIT-receptoraktivering resulterar i STAT3 Tyr 705- fosforylering via JAKl / 2. Efter STAT3-aktivering och nukleär translokation binds STAT3 till TGF-P1 ligandgenen och reglerar dess uttryck. Här demonstrerar vi den väsentliga rollen av ChIP-analys för identifiering av SMAD2-bindning till c-KIT-receptorligandpromotorn och för identifiering av signaltransducer (och) aktivator (av) transkription 3 (STAT3-bindning till TGF-P1- gen).

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">HepG2 cells</td>
			<td style="width:193px;">ATCC</td>
			<td style="width:119px;">HB-8065</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Hep3B cells</td>
			<td style="width:193px;">ATCC</td>
			<td>HB-8064</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">TGF-&#946;1</td>
			<td>R&#38;D Systems</td>
			<td>101-B1</td>
			<td>Used at a concentration of 10 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-SMAD2 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">5339</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-STAT3 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">4904</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">ChIP-IT Protein G Magnetic Beads</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">53033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Protease Inhibitor Cocktail</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">37490</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Micrococcal Nuclease</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">10011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GGAAGAGGATAAAGGACAAGCTG-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCAGCCAGCCACGTGATTGTC-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-CACTGATGTTAATGTTCAGC-3&#8217;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GCTCTAATTTAAACCTGGAGC-3&#8217;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GAGAGAGACGTGAGTGGCATGTT-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TAGCTTTCTCTGCCTTGGTCTCCCC-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GTACTGGGGGAGGAGCGGCATC-3&#8217;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCCACTGTCTGGAGAGAGGTGTGTC-3&#8217;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

analysis of the c-kit ligand promoter using chromatin immunoprecipitation

Multipla cellulära processer, inklusive DNA-replikation och reparation, DNA-rekombination och genuttryck, kräver interaktioner mellan proteiner och DNA. Därför reglerar DNA-proteininteraktioner multipla fysiologiska, patofysiologiska och biologiska funktioner, såsom celldifferentiering, cellproliferation, cellcykelstyrning, kromosomstabilitet, epigenetisk genreglering och celltransformation. I eukaryota celler interagerar DNA med histon- och nonhistonproteiner och kondenseras till kromatin. Flera tekniska verktyg kan användas för att analysera DNA-proteininteraktioner, såsom elektrofotografi (gel) Mobility Shift Assay (EMSA) och DNase I-fotavtryck. Dessa tekniker analyserar emellertid protein-DNA-interaktionen in vitro , inte inom det cellulära sammanhanget. Kromatinimmunutfällning (ChIP) är en teknik som fångar proteiner vid deras specifika DNA-bindningsställen, vilket möjliggör identifiering av DNA-proteininteraktionS inom deras kromatinkontext. Det görs genom fixering av DNA-proteininteraktionen, följt av immunutfällning av proteinet av intresse. Därefter karakteriserades den genomiska platsen som proteinet var bunden till. Här beskriver och diskuterar vi ChIP och demonstrerar dess analytiska värde för identifieringen av transformerande tillväxtfaktor-p (TGF-p) -inducerad bindning av transkriptionsfaktorn SMAD2 till SMAD-bindande element (SBE) inom promotorområdet hos tyrosin- Proteinkinas (c-KIT) -receptorligandstamcellsfaktor (SCF).

Bindningen av TGF-P1-liganden till dess kognitiva receptorkomplex resulterar i serin-fosforylering av SMAD2 / 3-transkriptionsfaktorer, följt av deras association med den gemensamma mediatorn, SMAD4. SMAD-komplexet translokerar till kärnan. TGF-p kan reglera gentranskription, antingen direkt via SMAD-bindning till SBE i inriktningsregioner hos målgenerna eller indirekt genom SMAD-reglerat uttryck av transkriptionella aktivatorer eller repressorer som därefter reglerar uttrycket av ge...

Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

DNA-protein interaktioner är väsentliga för flera biologiska processer. Under utvärderingen av cellulära funktioner är analysen av DNA-protein interaktioner oumbärlig för att förstå genreglering. Kromatinimmunutfällning (ChIP) är ett kraftfullt verktyg för att analysera sådana interaktioner in vivo.

Analys av c-KIT-ligand-promotorn med användning av kromatinimmunfällning

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

The overall goal of this chromatin immunoprecipitation procedure is to evaluate TGF beta 1-induced binding of the transcription factor SMAD2 to SMAD binding elements in the C kit promoter. This method can answer key questions in the field of cellular biology such as cytokine induced gene transcription. The main advantage of this technique is that it has the potential to demonstrate direct binding of transcription factors and other transcription regulatory proteins to their DNA binding sites in vivo.Grow the cells to between 70 and 80%confluence on ten centimeter tissue culture plates. Stimulate the cells per the experimental aims. Remove the tissue culture medium, and add five milliliters of fixation solution.Then, incubate the cells for 10 minutes at room temperature on a shaking platform. Remove the fixation solution, and wash the cells twice with 10 milliliters of ice cold one X PBS. Remove the one X PBS and add five milliliters of glycine stop fix solution.After incubating the cells for five minutes at room temperature on a shaking platform, remove the glycine stop fix solution and wash the cells twice with 10 milliliters of ice cold one X PBS. After removing the PBS, add two milliliters of cell scraping solution and harvest the cells using a cell scraper. Then, transfer the harvested cells to a conical tube on ice.Centrifuge the samples for 10 minutes at 600 times G, and four degrees celsius. Remove the supernatant, re-suspend the pellet in one milliliter of one X cell lysis buffer, and incubate on ice for 30 minutes. Once the pellet is re-suspended in the cell lysis buffer, transfer the cell suspension to an ice cold Dounce homogenizer, and Dounce for 10 strokes using a type B pestle for cell disruption.Then, transfer the cell suspension to a microcentrifuge tube, and centrifuge the sample for 10 minutes at 2, 400 times G, and four degrees celsius. Discard the supernatant and re-suspend the pellet in 350 microliters of nuclei digestion buffer. After incubating the samples for five minutes at 37 degrees celsius, add micrococcal nuclease and mix by vortexing.Incubate the samples at 37 degrees celsius for five to 20 minutes, mixing the samples every two minutes by inversion. The time in some concentration, if you're between cell lines, it might require optimization in case of suboptimal enzymatic shearing. An alternative shearing procedure using commercially available sonicare can be found in the text protocol.Following the incubation with the micrococcal nuclease, add seven microliters of ice cold 0.5 molar EDTA to stop the reaction. Centrifuge the samples with the sheared DNA for 10 minutes at 16, 200 times G and four degrees celsius. Transfer the sheared DNA containing supernatant to a fresh microcentrifuge tube.Aliquot 50 microliters into a separate microcentrifuge tube, to confirm the successful shearing of the DNA as described in the text protocol. Use the remaining volume immediately for immunoprecipitation or store it at minus 80 degrees celsius. Combine 10 to 25 micrograms of the sheared cross linked chromatin with 25 microliters of chromatin immunoprecipitation grade protein G magnetic beads.Then, add 10 microliters of immunoprecipitation dilution buffer, and one microliter of protease inhibitor cocktail. After four hours of incubation, add one to 10 micrograms of antibody to a 1.5 milliliter microcentrifuge tube. And add distilled water to a final volume of 100 microliters.Incubate the samples on an end-to-end rotator for four to 12 hours at four degrees celsius. Following incubation, place the tubes on a magnetic stand. Once the magnetic beads aggregate to the side of the tube, carefully remove and discard the supernatant.Wash the beads three times with 800 microliters of wash buffer one. For each wash, mix the beads by inverting the tube several times. Then, place the tube on the magnetic stand and carefully remove the wash buffer once the magnetic beads aggregate on the side of the tube.Next, wash the beads once with 800 microliters of wash buffer two using the same procedure. After washes, re-suspend the beads in 50 microliters of elution buffer and incubate the samples on an end-to-end rotator for 15 minutes at room temperature. Then, transfer the supernatant to a fresh tube with the aid of the magnetic stand.Add six microliters of five molar sodium chloride to reverse the cross linking followed by two microliters of proteinase K.After mixing the samples, incubate them for two hours at 65 degrees celsius. At this point, the samples can be directly analyzed for protein binding sites as described in the text protocol, or, can be stored at minus 20 degrees celsius. To confirm the TGF beta 1-induced binding of SMAD2 to the SCF promoter, the chromatin immunoprecipitation assay was performed.The TGF beta 1 treatment resulted in SMAD2 binding to the SCF promoter in human liver cancer lines, HepG2 and Hep3B. In the absence of TGF beta 1 stimulation, no SMAD binding was noted. As a positive control, for TGF beta 1-induced SMAD activation, chromatin immunoprecipitation for PAI-1 was performed.The PAI-1 promoter is a known target of TGF beta and SMAD. Specificity of the SMAD2 immunoprecipitation was confirmed through use of unspecific IgG antibodies. No SMAD2 binding to the SBE was noted after immunoprecipitation with IgG.To confirm the TGF Beta 1-induced binding of STAT3 to the TGF beta ligand gene, chromatin immunoprecipitation assays were performed using TGF beta 1 treated HepG2 and Hep3B cells. The TGF beta 1 treatment resulted in STAT3 binding to the second putative STAT3 binding site of the TGF beta gene, but not to the first one. In this example, the positive STAT3 binding to STB-2 serves as an internal positive control.Similar to the other chromatin immunoprecipitation experiment, STAT3 binding to the ST-2 was not seen after immunoprecipitation using IgG. Once mastered, this technique can be done in one or two days depending on the antibody if it is performed properly. While attempting this procedure, it's important to follow laboratory safety guidelines and be highly accurate at every step of the protocol.Precautions such as checking MSDS and wearing protective gear such as gloves, white coat and safety glasses should be taken while performing this procedure. After watching this video, you should have a good understanding of how to analyze protein binding to the specific DNA binding sequences. Following this protocol, other methods like promoter assays can be performed in order to confirm the functional relevance of protein DNA binding demonstrated by chromatin immunoprecipitation.

Research

JoVE Journal

Genetics

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Sekventiell Salt extraktioner för analys av Bulk kromatin bindande egenskaper av kromatin ändra komplex

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

Kromatinimmunutfällning (ChIP) i mus-T-celllinjer

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

Generation av infödda kromatin Immunoprecipitation sekvensering bibliotek för Nucleosome densitet analys

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Kromatin Immunoprecipitation (ChIP) protokoll för låg-överflöd embryonala prover

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Kromatin Immunoprecipitation (ChIP) Histon ändringar från Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

Arrangören Capture-C: Högupplösta, Genome-wide profilering av promotorn interaktioner

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Kromatin Immunoprecipitation av murina Brun fettvävnad

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

Kromatin immunoprecipitation analys med hjälp av Micrococcal nukleaser i däggdjursceller

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Upptäcka CsgD regulatoriska mål i salmonella biofilm med kromatin immunoprecipitation och högt dataflöde sekvensering (chip-SEQ)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Högupplöst kartläggning av protein-DNA-interaktioner i musstamcellsbaserade nervceller med chromatinimmunprecipitation-exonukleas (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...