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 brugen af hypoxi arbejdsstation. Dette arbejde blev støttet af følgende: Singapore Ministeriet for uddannelse, MOE 1T1-02/04 og MOE2015-T2-2-087 (til Y.A.), Lee Kong Chian School of Medicine, Nanyang Technological University starttilskud M4230003 (til P.O.B.), det svenske Forskningsråd, den Familie Erling Persson Foundation, Novo Nordisk Fonden, Stichting af Jochnick Foundation, den svenske Diabetes Association, Scandia Insurance Company, Diabetes forskning og Wellness Foundation, Kaj von Kantzow fond, den Strategisk forskningsprogram i Diabetes på Karolinska Institutet, ERC ERC-2013-AdG 338936-Betalmage, og Knut og Alice Wallenberg Foundation.

Denne protokoltjenesten, som bruger menneskelige embryonale nyre 293A (HEK293A) celle, s følger retningslinjerne i menneskelige videnskabsetisk Komité i Nanyang Technological University, Singapore.
1. induktion af hypoxi i HEK293A celler
<ol><li>Forberede fire 10 cm retter og frø 3-5 x 106 HEK293A celler pr. parabol i 10 mL Dulbeccos modificerede Eagle's medium (DMEM, 4,5 g/L glukose) suppleret med 10% føtal bovint serum (FBS), 2 mM L-glutamin, 110 mg/L natrium pyruvat, 100 U/...

<ol>
	<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=Hypoxia-inducible+factors+in+physiology+and+medicine.">Hypoxia-inducible factors in physiology and medicine.</a> Cell. 148 (3), 399-408 (2012).</li><li>Bartels, K., Grenz, A., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+inflammation+are+two+sides+of+the+same+coin.">Hypoxia and inflammation are two sides of the same coin.</a> Proc Natl Acad Sci U S A. 110 (46), 18351-18352 (2013).</li><li>Jiang, B. H., Rue, E., Wang, G. L., Roe, R., 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=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. L., Jiang, B. H., Rue, E. A., 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=Hypoxia-inducible+factor+1+is+a+basic-helix-loop-helix-PAS+heterodimer+regulated+by+cellular+O2+tension.">Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.</a> Proc Natl Acad Sci U S A. 92 (12), 5510-5514 (1995).</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=Cell-type-specific+regulation+of+degradation+of+hypoxia-inducible+factor+1+alpha:+Role+of+subcellular+compartmentalization.">Cell-type-specific regulation of degradation of hypoxia-inducible factor 1 alpha: Role of subcellular compartmentalization.</a> Mol Cell Biol. 26 (12), 4628-4641 (2006).</li><li>Depping, 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=Nuclear+translocation+of+hypoxia-inducible+factors+(HIFs):+involvement+of+the+classical+importin+alpha/beta+pathway.">Nuclear translocation of hypoxia-inducible factors (HIFs): involvement of the classical importin alpha/beta pathway.</a> Biochim Biophys Acta. 1783 (3), 394-404 (2008).</li><li>Wei, H., 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+induces+oncogene+yes-associated+protein+1+nuclear+translocation+to+promote+pancreatic+ductal+adenocarcinoma+invasion+via+epithelial-mesenchymal+transition.">Hypoxia induces oncogene yes-associated protein 1 nuclear translocation to promote pancreatic ductal adenocarcinoma invasion via epithelial-mesenchymal transition.</a> Tumour Biol. 39 (5), (2017).</li><li>Chang, H. 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=Hypoxia+promotes+nuclear+translocation+and+transcriptional+function+in+the+oncogenic+tyrosine+kinase+RON.">Hypoxia promotes nuclear translocation and transcriptional function in the oncogenic tyrosine kinase RON.</a> Cancer Res. 74 (16), 4549-4562 (2014).</li><li>Moriya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+nature+of+overexpression+experiments.">Quantitative nature of overexpression experiments.</a> Mol Biol Cell. 26 (22), 3932-3939 (2015).</li><li>Prelich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+overexpression:+Uses,+mechanisms,+and+interpretation.">Gene overexpression: Uses, mechanisms, and interpretation.</a> Genetics. 190 (3), 841-854 (2012).</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=A+Notch-independent+mechanism+contributes+to+the+induction+of+Hes1+gene+expression+in+response+to+hypoxia+in+P19+cells.">A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.</a> Exp Cell Res. 358 (2), 129-139 (2017).</li><li>Farris, M. H., Ford, K. A., Doyle, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+assays+for+detection+and+characterization+of+protein+antimicrobials.">Qualitative and quantitative assays for detection and characterization of protein antimicrobials.</a> J Vis Exp. (110), e53819 (2016).</li><li>Chilov, D., 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=Induction+and+nuclear+translocation+of+hypoxia-inducible+factor-1+(HIF-1):+heterodimerization+with+ARNT+is+not+necessary+for+nuclear+accumulation+of+HIF-1alpha.">Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha.</a> J Cell Sci. 112 (Pt 8), 1203-1212 (1999).</li><li>Yin, 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=Arylsulfonamide+KCN1+inhibits+in+vivo+glioma+growth+and+interferes+with+HIF+signaling+by+disrupting+HIF-1alpha+interaction+with+cofactors+p300/CBP.">Arylsulfonamide KCN1 inhibits in vivo glioma growth and interferes with HIF signaling by disrupting HIF-1alpha interaction with cofactors p300/CBP.</a> Clin Cancer Res. 18 (24), 6623-6633 (2012).</li><li>Holmquist-Mengelbier, L., 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=Recruitment+of+HIF-1alpha+and+HIF-2alpha+to+common+target+genes+is+differentially+regulated+in+neuroblastoma:+HIF-2alpha+promotes+an+aggressive+phenotype.">Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype.</a> Cancer Cell. 10 (5), 413-423 (2006).</li><li>Koh, M. Y., Powis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passing+the+baton:+The+HIF+switch.">Passing the baton: The HIF switch.</a> Trends Biochem Sci. 37 (9), 364-372 (2012).</li><li>Dumetz, A. C., Snellinger-O'brien, A. M., Kaler, E. W., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+protein+protein+interactions+in+salt+solutions+and+implications+for+protein+crystallization.">Patterns of protein protein interactions in salt solutions and implications for protein crystallization.</a> Protein Sci. 16 (9), 1867-1877 (2007).</li><li>Graven, K. K., Troxler, R. F., Kornfeld, H., Panchenko, M. V., Farber, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endothelial+cell+glyceraldehyde-3-phosphate+dehydrogenase+expression+by+hypoxia.">Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia.</a> J Biol Chem. 269 (39), 24446-24453 (1994).</li><li>Caradec, 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=Desperate+house+genes':+The+dramatic+example+of+hypoxia.">Desperate house genes': The dramatic example of hypoxia.</a> Br J Cancer. 102 (6), 1037-1043 (2010).</li></ol>

HIF-1 komplekset er en master regulator af cellulær ilt homøostase og regulerer et væld af gener involveret i forskellige cellulære adaptive responser til hypoxi. Identifikation af roman HIF-1 interagerende proteiner er vigtige for forståelsen af hypoksiske signaltransduktion. Co-immunoprecipitation eksperimenter er almindeligt anvendt til PPI undersøgelser for at afgrænse cellulære signaltransduktionsveje. Men protein overekspression er stadig udbredt og dette kan føre til eksperimentelle artefakter. Derudover ...

Iltsvind opstår, når utilstrækkelig ilt tilføres til celler og væv i kroppen. Det spiller en afgørende rolle i forskellige fysiologiske og patologiske processer såsom stamcelle differentiering, betændelse og cancer1,2. Hypoxi-inducerbar faktorer (HIFs) fungere som heterodimers består af en ilt-regulerede α-subunit og en constitutively udtrykt β-underenheden også kendt som ARNT3. Tre isoformer af HIF-α underenheder (HIF-1α, HIF-2α og HIF-3α) og tre HIF-β underenheder (ARNT/HIF-1β, ARNT2 og ARNT3) er blevet identificeret til dato. HIF-1α og ARNT udtrykkes allestedsnærværende, mens HIF-2α, HIF-3α, ARNT2 og ARNT3 har mere begrænset udtryk mønstre4. HIF-1 protein kompleks er den centrale regulator af hypoxi svar. Hypoksiske betingelser HIF-1α bliver stabiliseret, derefter translocates til kernen og dimerizes med ARNT5. Senere, dette kompleks binder sig til specifikke nukleotider kendt som hypoxi lydhør elementer (HREs) og regulerer udtryk for target gener involveret i forskellige processer herunder angiogenese, erythropoiesen og glykolyse6. Ud over denne "canonical" svar, hypoxi signalvejen er også kendt at krydstale med flere cellulære reaktion signalering veje som Notch og nuklear Factor-kappa B (NF-κB)7,8,9.
Identifikation af roman HIF-1 interagerende proteiner er vigtige for en bedre forståelse af hypoxi signalering pathway. I modsætning til ARNT, der er ufølsom over for iltindholdet og constitutively udtrykt, er HIF-1α protein niveauer stramt reguleret af cellulær ilt niveauer. På normoxia (21% ilt) er HIF-1α proteiner hurtigt forringet10,11. HIF-1α kort halveringstid på normoxia præsenterer specifikke tekniske udfordringer for påvisning af protein fra celle ekstrakter og for identifikation af HIF-1α-interagere proteiner. Desuden translocate flere transskriptionsfaktorer herunder HIF-1 kompleks ind i kernen under hypoksiske betingelser12,13,14. De fleste af de nuværende metoder, der anvendes for PPI undersøgelser udføres ved hjælp af ikke-fysiologisk overekspression af proteiner. Sådanne protein overekspression er blevet rapporteret at forårsage forskellige cellulære defekter gennem flere mekanismer, herunder ressource overbelastning, støkiometriske ubalance, promiskuøs interaktioner og pathway graduering15,16. Med hensyn til PPI undersøgelser, kan protein overekspression føre til falsk positiv, eller sågar falske negative, resultater afhængigt af protein egenskaber og funktioner af overexpressed proteiner. De nuværende metoder for PPI undersøgelser skal derfor ændres for at afsløre de fysiologisk relevante PPI hypoksiske betingelser. Vi har tidligere vist samspillet mellem HIF-1 og Ets familie transkriptionsfaktor GA-bindende protein (GABP) i hypoksiske P19 celler, som bidrager til svar af Hes1 initiativtageren til hypoxi17. Her, beskriver vi en co-immunoprecipitation protokol for at studere PPI mellem endogene nukleare proteiner hypoksiske betingelser. Samspillet mellem HIF-1α og ARNT er vist som en proof of concept. Denne protokol er egnet til viser samspillet mellem transkriptionsfaktorer og transcriptional Co regulatorer hypoksiske betingelser, herunder men ikke begrænset til identifikation af HIF-1 interagerende proteiner.

<table border="0" cellpadding="0" cellspacing="0" width="803">
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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>
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co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

Lavt iltindhold (hypoksi) udløse en bred vifte af adaptive responser med hypoxi-inducerbar faktor 1 (HIF-1) kompleks som en master regulator. HIF-1 består af en heterodimerisk ilt-regulerede α-underenhed (HIF-1α) og constitutively udtrykt β-underenheden (HIF-1β) også kendt som aryl kulbrinte receptor nukleare translocator (ARNT), regulere gener involveret i forskellige processer herunder angiogenese , erythropoiesen og glykolyse. Identifikation af HIF-1 interagerende proteiner er nøglen til forståelsen af hypoxi signalering pathway. Ud over regulering af HIF-1α stabilitet udløser hypoxi også nukleare omplantning af mange transskriptionsfaktorer herunder HIF-1α og ARNT. Navnlig, er de fleste af de nuværende metoder, der anvendes til at studere disse protein-protein interaktioner (ppi) baseret på systemer hvor protein niveauer er kunstigt forøget gennem protein overekspression. Protein overekspression ofte fører til ikke-fysiologiske resultater som følge af tidsmæssige og rumlige artefakter. Her beskriver vi en modificeret co-immunoprecipitation protokol efter hypoxi behandling ved hjælp af endogene nukleare proteiner, og som en proof of concept viser samspillet mellem HIF-1α og ARNT. I denne protokol, de hypoksiske celler blev høstet hypoksiske betingelser og den Dulbecco Phosphate-Buffered saltvand (DPBS) wash buffer var også pre afbalancerede hypoksiske betingelser før skik at afbøde protein nedbrydning eller protein kompleks dissociation under reoxygenation. Derudover var de nukleare fraktioner efterfølgende udvundet for at koncentrere sig og stabilisere endogene nukleare proteiner og undgå eventuelle falske resultater ses ofte under protein overekspression. Denne protokol kan bruges til at påvise endogene og indfødte interaktioner mellem transkriptionsfaktorer og transcriptional Co regulatorer hypoksiske betingelser.

For at vurdere den cellulære reaktion på hypoxi, udtrykket niveauer og subcellulært lokalisering af komponenterne i HIF-1 komplekse følgende hypoxi behandlingen blev undersøgt. HEK293A celler blev kulturperler hypoksiske betingelser for 4 h eller holdes på normoxia som kontrol. HIF-1α og ARNT protein niveauer blev undersøgt i hele cellen eller nukleare/cytoplasmatisk uddrag af vestlige skamplet. Som forventede, samlede HIF-1α niveauer var upregulated af hypoxi, mens ARNT niveauer...

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 protokol for at studere protein-protein interaktioner mellem endogene nukleare proteiner hypoksiske betingelser. Denne metode er velegnet til demonstration af vekselvirkninger mellem transkriptionsfaktorer og transcriptional Co lovgivere på hypoxi.

Co-immunoprecipitation analyse ved hjælp af endogene nukleare proteiner fra celler kulturperler hypoksiske betingelser

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

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Biology

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