Name: co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
Uploaded: 2018-08-02T14:30:00
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Wij danken Assoc. Prof. Sin Tiong Ong voor het gebruik van het werkstation van hypoxie. Dit werk werd ondersteund door de volgende: Singapore ministerie van onderwijs, MOE 1T1-02/04 en MOE2015-T2-2-087 (naar Y.A.), Lee Kong Chian School of Medicine, Nanyang Technological University start-up subsidie M4230003 (P.O.B.), de Zweedse Raad voor onderzoek, de Familie Erling Persson Stichting, de Novo Nordisk Stichting, de Stichting af Jochnick Foundation, de Zweedse vereniging van de Diabetes, de verzekeringsmaatschappij Scandia, het onderzoek naar Diabetes en Wellness Foundation, ligplaats von Kantzow van de Stichting, de Strategische onderzoeksprogramma in Diabetes op Karolinska Institutet, de ERC ERC-2013-AdG-338936-Betalmage, en de Knut en Alice Wallenberg Foundation.

Dit gedeelte van het protocol, die gebruik maakt van menselijke embryonale nier 293A (HEK293A) de cel, s volgt de richtsnoeren van de Commissie van de ethiek van de menselijke onderzoek in Nanyang Technological University, Singapore.
1. de inductie van hypoxie in HEK293A cellen
<ol><li>Bereiden vier 10 cm schotels en zaad 3 – 5 x 106 HEK293A cellen per schotel in 10 mL Dulbecco van Eagle's medium (DMEM, 4.5 g/L glucose bewerkt) aangevuld met 10% foetale runderserum (FBS), 2 mM L...

<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. 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De HIF-1-complex is een meester regulator van cellulaire zuurstof homeostase en regelt een overvloed van genen die betrokken zijn bij verschillende cellulaire adaptieve reacties op hypoxie. Identificatie van nieuwe HIF-1 interacterende eiwitten is belangrijk voor het begrijpen van hypoxische signaaltransductie. Co-immunoprecipitation experimenten worden vaak gebruikt voor PPIs studies af te bakenen Cellulaire signaaltransductie trajecten. Echter, eiwit overexpressie is nog steeds veel gebruikt en dit kan leiden tot exper...

Hypoxie treedt op wanneer onvoldoende zuurstof aan de cellen en weefsels van het lichaam wordt geleverd. Het speelt een cruciale rol in verschillende fysiologische en pathologische processen zoals stamcel differentiatie, ontsteking en kanker1,2. Hypoxie-afleidbare factoren (HIFs) fungeren als heterodimers samengesteld uit een zuurstof-gereglementeerde α subeenheid en een constitutively uitgedrukt β-subunit ook bekend als ARNT3. Drie isoforms van de HIF-α-subunits (HIF-1α, HIF-2α en HIF-3α) en drie HIF-β-subunits (ARNT/HIF-1β, ARNT2 en ARNT3) zijn tot nu toe geïdentificeerd. HIF-1α en ARNT worden overal uitgedrukt, overwegende dat de HIF-2α HIF-3α, ARNT2 en ARNT3 hebben meer expressie patronen4beperkt. De HIF-1 eiwit complex is de belangrijke regulator van de reactie van hypoxie. Hypoxische omstandigheden HIF-1α wordt gestabiliseerd, vervolgens translocates op de nucleus en dimerizes met ARNT5. Vervolgens dit complex wordt gebonden aan bepaalde nucleotiden hypoxie responsieve elementen (HREs) genoemd en regelt de uitdrukking van de doelgenen die betrokken zijn bij verschillende processen, met inbegrip van angiogenese, Erytropoëse en glycolyse6. Naast deze "canonieke" reactie, de signalering traject van hypoxie is ook bekend Overspraak met meerdere cellulaire respons op de signalering van studierichtingen zoals de Inkeping en Nuclear Factor-kappa B (NF-recombination)7,8,9.
De identificatie van roman HIF-1 interacterende eiwitten is belangrijk voor een beter begrip van de signalering traject hypoxie. In tegenstelling tot ARNT, die ongevoelig voor zuurstofniveaus en constitutively uitgedrukt is, worden de HIF-1α eiwitniveaus strak gereguleerd door cellulaire zuurstofniveaus. Normoxia (21% zuurstof) zijn HIF-1α proteïnen snel aangetaste10,11. De korte halfwaardetijd van HIF-1α op normoxia biedt specifieke technische uitdagingen voor de detectie van het eiwit uit cel extracten, alsook voor de identificatie van eiwitten HIF-1α-interactie. Bovendien translocate verschillende transcriptiefactoren met inbegrip van die van het complex HIF-1 in de kern onder hypoxische voorwaarden12,13,14. De meeste van de huidige methoden voor PPI studies worden uitgevoerd met behulp van niet-fysiologische overexpressie van eiwitten. De overexpressie van dergelijke eiwitten is gemeld aan de verschillende cellulaire defecten veroorzaken door meerdere mechanismen, met inbegrip van resource overbelasting, stoichiometrische onbalans, promiscue interacties en traject modulatie15,16. In termen van PPI studies, kan eiwit overexpressie leiden tot valse positieve, of zelfs valse negatief, resultaten, afhankelijk van de eigenschappen van de eiwitten en de functies van de overexpressie eiwitten. Daarom moeten de huidige methoden voor PPI studies worden gewijzigd om te onthullen het fysiologisch relevante PPIs onder hypoxische voorwaarden. Wij hebben eerder aangetoond dat de interactie tussen HIF-1 en de familie transcriptiefactor Ets GA-bindend-proteïne (GABP) in hypoxische P19 cellen, die tot de reactie van de Hes1 -promotor op hypoxie17 bijdraagt. Hier beschrijven we een co-immunoprecipitation protocol om te studeren van PPIs tussen endogene nucleaire eiwitten hypoxische omstandigheden. De interactie tussen HIF-1α en ARNT wordt weergegeven als een proof of concept. Dit protocol is geschikt voor het aantonen van de interacties tussen transcriptiefactoren en transcriptionele co regelgevers hypoxische voorwaarden, met inbegrip van maar niet beperkt tot de identificatie van HIF-1 interacterende eiwitten.

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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">
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co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

Lage zuurstofniveaus (hypoxie) leiden tot een verscheidenheid van adaptieve reacties met de hypoxie-afleidbare factor 1 (HIF-1) complexe fungeert als een meester regulator. HIF-1 bestaat uit een heterodimeric zuurstof-gereglementeerde α subeenheid (HIF-1α) en constitutively uitgedrukt β subeenheid (HIF-1β) ook bekend als aryl koolwaterstof receptor nucleaire translocator (ARNT), regulering van de genen die betrokken zijn bij verschillende processen, met inbegrip van angiogenese , Erytropoëse en glycolyse. De identificatie van HIF-1 interacterende eiwitten is de sleutel tot het begrip van de signalering traject hypoxie. Naast de regulering van de HIF-1α stabiliteit leidt hypoxie ook tot de nucleaire translocatie van vele transcriptiefactoren waaronder HIF-1α en ARNT. Met name, zijn de meeste van de huidige methoden gebruikt bij het bestuderen van deze eiwit-eiwitinteractie (PPIs) gebaseerd op systemen waar eiwitniveaus kunstmatig worden verlengd door middel van eiwit overexpressie. Eiwit overexpressie vaak leidt tot niet-fysiologische resultaten die voortvloeien uit de temporele en ruimtelijke artefacten. Hier beschrijven we een gemodificeerde co-immunoprecipitation protocol na hypoxie behandeling met behulp van endogene nucleaire eiwitten, en als een proof of concept, om te laten zien van de interactie tussen HIF-1α en ARNT. In dit protocol, de hypoxische cellen zijn geoogst hypoxische omstandigheden en van de Dulbecco zoute Phosphate-Buffered (DPBS) was buffer was ook vooraf equilibrated hypoxische voorwaarden vóór gebruik ter vermindering van de aantasting van het proteïne of eiwit complex Dissociatie tijdens heroxygenatie. Daarnaast werden de nucleaire breuken vervolgens gehaald om te concentreren en het stabiliseren van de endogene nucleaire eiwitten en het voorkomen van mogelijke valse resultaten vaak gezien tijdens eiwit overexpressie. Dit protocol kan worden gebruikt om aan te tonen van endogene en inheemse interacties tussen transcriptiefactoren en transcriptionele co regelgevers hypoxische omstandigheden.

Om te evalueren van de cellulaire reactie op hypoxie, de expressie niveaus en subcellular localisatie van de componenten van de HIF-1 complexe volgende hypoxie behandeling werden onderzocht. HEK293A cellen werden gekweekte hypoxische voorwaarden voor 4 uur of als besturingselementen op normoxia gehouden. HIF-1α en ARNT eiwitniveaus werden onderzocht in hele cel of nucleaire/cytoplasmatische uittreksels door westelijke vlek. Zoals verwachte, totale HIF-1α niveaus upregulated door hypoxie...

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

Hier beschrijven we een co-immunoprecipitation protocol om te bestuderen van de eiwit-eiwit interactie tussen endogene nucleaire eiwitten hypoxische omstandigheden. Deze methode is geschikt voor de demonstratie van de interacties tussen transcriptiefactoren en transcriptionele co regelgevers op hypoxie.

Co-immunoprecipitation Assay met behulp van endogene nucleaire eiwitten uit cellen gekweekt onder hypoxische voorwaarden

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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Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

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