Name: co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
Uploaded: 2018-08-02T14:30:00
Description: Watch this Scientific Journal Video about Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions at JoVE.com

Vi takker Assoc. Prof synd Tiong Ong for bruk av hypoksi arbeidsstasjonen. Dette arbeidet ble støttet av følgende: Singapore Utdanningsdepartementet, MOE 1T1-02/04 og MOE2015-T2-2-087 (til Y.A.), Lee Kong Chian School of Medicine, Nanyang Technological University oppstart grant M4230003 (til P.O.B.), den svenske Research Council, den Familie Erling Persson Foundation, Novo Nordisk stiftelsen Stichting af Jochnick Foundation, den svenske Diabetes Association, Scandia forsikringsselskapet, Diabetes Research og velvære Foundation, køye von Kantzow Foundation, den Strategisk forskningsprogram i Diabetes ved Karolinska Institutet, ERC ERC-2013-AdG 338936-Betalmage, og Knut og Alice Wallenberg Foundation.

Denne protokollen delen, som bruker menneskelige embryonale nyre 293A (HEK293A) celle, s følger retningslinjene for menneskelig forskning etikk i Nanyang Technological University, Singapore.
1. innledning av oksygenmangel i HEK293A celler
<ol><li>Forberede fire 10 cm retter og frø 3 – 5 x 106 HEK293A celler per fat i 10 mL Dulbeccos endret Eagle's medium (DMEM, 4,5 finans glukose) supplert med 10% fosterets bovin serum (FBS), 2 mM L-glutamin, 110 mg/L natrium pyruvate, 100 U/m...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization,+DNA+binding,+and+transactivation+properties+of+hypoxia-inducible+factor+1.">Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.</a> J Biol Chem. 271 (30), 17771-17778 (1996).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1:+mediator+of+physiological+and+pathophysiological+responses+to+hypoxia.">HIF-1: mediator of physiological and pathophysiological responses to hypoxia.</a> J Appl Physiol. 88 (4), 1474-1480 (2000).</li><li>Kallio, P. J., 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=Signal+transduction+in+hypoxic+cells:+Inducible+nuclear+translocation+and+recruitment+of+the+CBP/p300+coactivator+by+the+hypoxia-inducible+factor-1alpha.">Signal transduction in hypoxic cells: Inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha.</a> EMBO J. 17 (22), 6573-6586 (1998).</li><li>Ke, Q., Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-1+(HIF-1).">Hypoxia-inducible factor-1 (HIF-1).</a> Mol Pharmacol. 70 (5), 1469-1480 (2006).</li><li>Gustafsson, M. V., 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=Hypoxia+requires+notch+signaling+to+maintain+the+undifferentiated+cell+state.">Hypoxia requires notch signaling to maintain the undifferentiated cell state.</a> Dev Cell. 9 (5), 617-628 (2005).</li><li>Zheng, X., 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=Interaction+with+factor+inhibiting+HIF-1+defines+an+additional+mode+of+cross-coupling+between+the+Notch+and+hypoxia+signaling+pathways.">Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways.</a> Proc Natl Acad Sci U S A. 105 (9), 3368-3373 (2008).</li><li>D'Ignazio, L., Bandarra, D., Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+and+HIF+crosstalk+in+immune+responses.">NF-kappaB and HIF crosstalk in immune responses.</a> FEBS J. 283 (3), 413-424 (2016).</li><li>Wang, G. 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HIF-1 komplekset er en master regulator mobilnettet oksygen homeostasis og regulerer en mengde tilblivelse involvert inne forskjellige mobilnettet adaptive svar til hypoksi. Identifikasjon av romanen HIF-1 samspill proteiner er viktig for forståelsen av hypoxic signaltransduksjon. Co-immunoprecipitation eksperimenter brukes vanligvis for PPIs studier for å avgrense mobilnettet signaltransduksjon trasé. Men protein overuttrykte er fremdeles mye brukt, og dette kan føre til eksperimentelle gjenstander. I tillegg HIF-1?...

Hypoxia oppstår når utilstrekkelig oksygen angis til celler og vev i kroppen. Det spiller en avgjørende rolle i ulike fysiologiske og patologiske prosesser som stamcelleforskningen differensiering, betennelse og kreft1,2. Hypoksi-induserbart faktorer (HIFs) fungerer som heterodimerer består av en oksygen regulert α delenhet og en constitutively uttrykt β delenhet ARNT3også. Tre isoformene HIF-α underenheter (HIF-1α, HIF-2α og HIF-3α) og tre HIF-β underavdelinger (ARNT/HIF-1β, ARNT2 og ARNT3) har blitt identifisert hittil. HIF-1α og ARNT uttrykkes overalt, mens HIF-2α, HIF-3α, ARNT2 og ARNT3 har mer begrenset uttrykk mønstre4. HIF-1 proteinet er viktig regulator av hypoksi respons. Under hypoxic forhold, HIF-1α blir stabilisert, translocates til kjernen og dimerizes med ARNT5. Deretter Dette komplekset binder til spesifikke nukleotider kjent som hypoksi forståelsesfull elementer (HREs) og regulerer uttrykket av målet gener involvert i ulike prosesser inkludert angiogenese, erythropoiesis og Glykolysen6. I tillegg denne "kanoniske" respons, hypoksi signalveien kalles også å crosstalk med flere cellulær respons signalnettverk trasé som hakk og kjernefysiske faktor-kappa B (NF-κB)7,8,9.
Identifikasjon av romanen HIF-1 samspill proteiner er viktig for en bedre forståelse av hypoksi signalveien. I motsetning til ARNT, som ufølsom for oksygen og constitutively uttrykt, er HIF-1α protein nivå tett regulert av mobilnettet oksygennivået. På normoxia (21% oksygen) er HIF-1α proteiner raskt dårligere10,11. Den korte halveringstiden av HIF-1α på normoxia presenterer bestemte tekniske utfordringer for påvisning av protein fra cellen ekstrakter og identifikasjon av HIF-1α-samspill proteiner. Videre translocate flere transkripsjonsfaktorer inkludert de av HIF-1 komplekset i kjernen under hypoxic forhold12,13,14. De fleste av dagens metoder brukes for PPI studier utføres ved hjelp av ikke-fysiologiske overuttrykte proteiner. Slike protein overuttrykte har blitt rapportert til forskjellige mobilnettet defekter gjennom flere mekanismer, inkludert ressurs overbelastning, stoichiometric ubalanse, promiskuøse interaksjoner og veien modulering15,16. I PPI studier, kan protein overuttrykte føre til falsk positiv eller selv falsk negativt, resultater avhengig av protein egenskapene og funksjonene til overexpressed proteiner. Gjeldende metoder for PPI studier har derfor, for å avsløre de fysiologisk relevante PPIs under hypoxic forhold. Vi har tidligere vist samspillet mellom HIF-1 og Ets familie transkripsjon faktoren GA-bindende protein (GABP) i hypoxic P19 celler, noe som bidrar til responsen til Hes1 promoter hypoksi17. Her beskriver vi en co-immunoprecipitation-protokoll for å studere PPIs mellom endogene kjernefysiske proteiner under hypoxic forhold. Samspillet mellom HIF-1α og ARNT vises som et bevis på konseptet. Denne protokollen er egnet for å vise interaksjonen mellom transkripsjonsfaktorer og transcriptional co regulatorer under hypoxic forhold, inkludert men ikke begrenset til identifisering av HIF-1 samspill proteiner.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
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			<td height="21" style="height:21px;width:279px;">Material</td>
			<td style="width:161px;"></td>
			<td style="width:135px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1.0 M Tris-HCl Buffer, pH 7.4&#160;</td>
			<td style="width:161px;">1st BASE</td>
			<td style="width:135px;">1415</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Protein A/G Sepharose beads</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:135px;">ab193262</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Natural Mouse IgG protein</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:135px;">ab198772</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">EDTA</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610729</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">2x Laemmli Sample Buffer</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610737</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">2-Mercaptoethanol</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610710</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nitrocellulose Membrane &#160;&#160;</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1620112</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Blotting-Grade Blocker</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1706404</td>
			<td style="width:229px;">Non-fat dry milk for western blotting applications</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris Buffered Saline (TBS)</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1706435</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10% Tween 20</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610781</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris/Glycine/SDS</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610732</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris/Glycine Buffer&#160;</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610771</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Precision Plus Protein Dual Color Standards</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610374</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">7074</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anti-mouse IgG, HRP-linked Antibody&#160;</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">7076</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SignalFire ECL Reagent</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">6883</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">21-030-CV</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:135px;">52332</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ARNT/HIF-1 beta Antibody&#160;</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NB100-124&#160;</td>
			<td style="width:229px;">Concentration: 1.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">HIF-1 alpha Antibody</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NB100-479</td>
			<td style="width:229px;">Concentration: 1.0 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">YY1 Antibody</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NBP1-46218</td>
			<td style="width:229px;">Concentration: 0.2 mg/mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Qproteome Nuclear Protein Kit</td>
			<td style="width:161px;">Qiagen</td>
			<td style="width:135px;">37582</td>
			<td style="width:229px;">Lysis buffer NL and Extraction Buffer NX1 are provied in the kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">GAPDH Antibody</td>
			<td style="width:161px;">Santa Cruz</td>
			<td style="width:135px;">sc-47724</td>
			<td style="width:229px;">Concentration: 0.2 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol (&#8805;99%)</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Potassium chloride</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RIPA buffer</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">R0278</td>
			<td></td>
		</tr>
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co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

Lavt oksygennivå (hypoksi) utløse en rekke adaptive svar med hypoksi-induserbart faktor 1 (HIF-1) kompleks som en master regulator. HIF-1 består av en heterodimeric oksygen regulert α undergruppe (HIF-1α) og constitutively uttrykt β delenhet (HIF-1β) også kjent som aryl hydrokarbon reseptor kjernefysiske translocator (ARNT), regulerer gener involvert i ulike prosesser inkludert angiogenese , erythropoiesis og Glykolysen. Identifikasjon av HIF-1 samspill proteiner er nøkkelen til forståelsen av hypoksi signalveien. I tillegg til regulering av HIF-1α stabilitet utløser hypoksi også kjernefysiske translokasjon av mange transkripsjonsfaktorer inkludert HIF-1α og ARNT. Spesielt, er de fleste av dagens metoder brukes til å studere slik protein-protein interaksjoner (PPIs) basert på systemer der protein nivå er kunstig økt gjennom protein overuttrykte. Protein overuttrykte ofte fører til ikke-fysiologiske resultater som følge av timelige og romlig gjenstander. Her beskriver vi en modifisert co-immunoprecipitation protokollen etter hypoksi behandlingssøkende endogene kjernefysiske proteiner, og som et bevis på konseptet, å vise interaksjonen mellom HIF-1α og ARNT. I denne protokollen, hypoxic cellene ble høstet under hypoxic forhold og Dulbecco's Phosphate-Buffered saltvann (DPBS) vaskebuffer var også pre equilibrated hypoxic forhold før behandling å begrense protein fornedrelse eller protein kompleks dissosiasjon under reoxygenation. I tillegg kjernefysiske fraksjoner ble senere trukket ut å konsentrere seg og stabilisere endogene kjernefysiske proteiner og unngå mulige falske resultater ofte sett under protein overuttrykte. Denne protokollen kan brukes å demonstrere endogene og native interaksjoner mellom transkripsjonsfaktorer og transcriptional co regulatorer under hypoxic forhold.

For å vurdere i cellulær respons til hypoksi, uttrykket nivåer og subcellular lokalisering av komponentene i HIF-1 komplekse følgende hypoksi behandling ble undersøkt. HEK293A celler ble kultivert hypoxic vilkår 4 h eller holdt på normoxia kontroller. HIF-1α og ARNT protein nivå ble undersøkt i hele cellen eller kjernefysiske/cytoplasmatiske ekstrakter ved western blot. Som forventet, total HIF-1α nivåer var upregulated av hypoksi, mens ARNT nivåer i totale mobilnettet lysate...

Watch this Scientific Journal Video about Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions at JoVE.com

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Her beskriver vi en co-immunoprecipitation-protokoll for å studere protein-protein interaksjoner mellom endogene kjernefysiske proteiner under hypoxic forhold. Denne metoden er egnet for demonstrasjon av samspillet mellom transkripsjonsfaktorer og transcriptional co regulatorer på hypoksi.

Co-immunoprecipitation analysen bruke endogene kjernefysiske proteiner fra celler kultivert Hypoxic vilkår

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Research

JoVE Journal

Biology

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