Name: probing for mitochondrial complex activity in human embryonic stem cells
Uploaded: 2008-06-17T17:21:00
Description: Watch this Scientific Journal Video about Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells at JoVE.com

<p> Menselijke embryonale stamcellen studies in de Teitell lab worden momenteel ondersteund door een Californische Instituut voor Regeneratieve Geneeskunde (CIRM) Seed Grant RS1-00313. Wij danken dr. Carla Koehler (UCLA) en leden van het Koehler laboratorium voor het verstrekken van advies en inzicht in deze en aanvullende studies. </p>

<p class="jove_title"> Hoge resolutie Clear native Gel Electophoresis (hrCNE) voor mitochondriële complex In-Gel Activity Assays </p><p class="jove_content"> Menselijke embryonale stamcellen (hESC &#39;s) werden gehouden op een monolaag van ˠ-bestraalde of mitomycine C-behandelde muis embryonale fibroblasten (MEF), daarna overgestapt naar groei op Matrigel gedurende 5 dagen in de MEF-geconditioneerd medium met behulp van standaard voorwaarden. Voor het verwijderen van verontreiniging MEFs, werden hESC &#39;s opnieuw uitgeplaat op Matrigel en ...

<ol>
	<li>Wittig, I., Braun, H. -. P., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> <em style='font-style: italic'>Nature Protocols</em>. <b>1</b>, 418-428 (2006).</li><li>Wittig, I., Karas, M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Clear+Native+Electrophoresis+for+In-gel+Functional+Assays+and+Fluorescence+Studies+of+Membrane+Protein+Complexes.++6.">High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes.  6.</a> <em style='font-style: italic'>Molecular &amp; Cellular Proteomics</em>. <b>6</b>, 1215-1225 (2007).</li><li>Wittig, I., Carrozzo, R., Santorelli, F. M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines.">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> <em style='font-style: italic'>Electrophoresis</em>. <b>28</b>, 3811-3820 (2007).</li><li>Castle, J. D., Coligan, J. E., Dunn, B. M., Speicher, D. W., Wingfield, P. T. <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+Organelles+from+Mammalian+Cells.">Purification of Organelles from Mammalian Cells.</a> <em style='font-style: italic'>Current Protocols in Protein Science</em>. <b>4.2</b>, 4.2.1-4.2.57 (2007).</li></ol>

<p> In deze video, hebben we beschreven de extractie van de mitochondriale eiwitcomplexen uit menselijke embryonale stamcellen, hun scheiding door de hoge resolutie heldere inheemse gelelektroforese (hrCNE), en de daaropvolgende analyse door in-gel katalytische activiteit assays. hrCNE lost mitochondriale membraan eiwit complexen met een resolutie vergelijkbaar met die van autochtone blauwe gel elektroforese en is superieur voor in-gel-activiteit assays. Deze techniek kan niet alleen gebruikt ter mitochondriale ademhaling complexen IV te kwanti...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>0.5M EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP120-1</td>
<td>adjust pH to 8.0 with NaOH </td>
</tr>
<tr>
<td>1M NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014-500MG</td>
<td>&nbsp;</td>
<tr>
<td>1M Sodium succinate</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S413-500</td>
<td>&nbsp;</td>
<tr>
<td>0.1M Sodium phosphate buffer pH 7.2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP332-1 and BP329-1</td>
<td>Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C </td>
</tr>
<tr>
<td>1M Tricine, pH 7.0 (adjusted with NaOH)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>9010</td>
<td>store at 4 C </td>
</tr>
<tr>
<td>1M imidazole/HCl, pH 7.0</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-2399</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>1M Tris-HCl, pH 7.4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP153-1</td>
<td>&nbsp;</td>
<tr>
<td>20% (wt/vol) dodecyl-b-D-maltoside (DDM)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44205-500MG</td>
<td>Dissolved in water. Store 1 ml aliquots at &#x2013;20 C.</td>
</tr>
<tr>
<td>10% (wt/vol) digitonin (>50% purity, used without recrystallization)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>37008-1G</td>
<td>Dissolved in water. Store 0.1&#x2013;1 ml aliquots at &#x2013;20 C. Should be warmed up to 95 C before use.</td>
</tr>
<tr>
<td>2M 6-aminohexanoic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>07260-100G</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>10x Ponceau S/glycerol stock solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3504 and G6279-1L</td>
<td>0.1% Ponceau S, 50% glycerol, (wt/vol) in water.</td>
</tr>
<tr>
<td>10% w/v sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Fluka </td>
<td>30970-25G</td>
<td>Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.</td>
</tr>
<tr>
<td>250 mM phenazine methasulfate (PMS)</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>P0083</td>
<td>Dissolved in DMSO and aliquoted. Store at &#x2013;20 C.</td>
</tr>
<tr>
<td>Nitro-blue tetrazolium (NBT)</td>
<td>&nbsp;</td>
<td>Amresco </td>
<td>0329-1G</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride (DAB)</td>
<td>&nbsp;</td>
<td>TCI America </td>
<td>D0078</td>
<td>&nbsp;</td>
<tr>
<td>Pb(NO3)2</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>L62-100</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP213-1</td>
<td>&nbsp;</td>
<tr>
<td>Tris base </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP152-10</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>G45-212</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2019;-triphosphate disodium salt (ATP)</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2383-5G</td>
<td>Grade I, minimum 99%</td>
</tr>
<tr>
<td>beta-nicotineamide adenine dinucleotide (NADH) reduced form </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>N-8129</td>
<td>&nbsp;</td>
<tr>
<td>Cytochrome c From horse heart</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C2506-250MG</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP220-212</td>
<td>&nbsp;</td>
<tr>
<td>100x protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P8340</td>
<td>Store at &#x2013; 20 C.</td>
</tr>
<tr>
<td>Dimethyl sulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D128-500</td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue Stain 0.4%</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>10x Trypsin (0.5%)</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>17075-029</td>
<td>&nbsp;</td>		</tbody>
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probing for mitochondrial complex activity in human embryonic stem cells

<p class="jove_content">Mitochondriën zijn cytoplasmatische organellen die een primaire rol in de cellulaire metabolisme en homeostase hebben, de regulatie van de cel signaleringsnetwerk, en geprogrammeerde celdood. Mitochondria produceren ATP, reguleren van de cytoplasmatische redox staat en Ca2 + saldo, catabolize vetzuren, synthese van heem, nucleotiden, steroïde hormonen, aminozuren, en helpen monteren ijzer-zwavel clusters in eiwitten. Mitochondriën hebben ook een essentiële rol in de productie van warmte. Mutaties van het mitochondriaal genoom oorzaak verschillende soorten van de menselijke aandoening. De accumulatie van mtDNA mutaties correleert met het ouder worden en is vermoedelijk een belangrijke rol in de ontwikkeling van kanker te hebben. Door hun uiterst belangrijke rol in alle celtypes, moet de functie van mitochondriën ook kritisch zijn voor stamcellen. Belangrijke vooruitgang geboekt in onze kennis van stamcellen levensvatbaarheid, proliferatie en differentiatie capaciteit. Maar de functionele activiteit van stamcellen, in het bijzonder hun energie-status, was nog niet in detail bestudeerd. Bijna niets is bekend over de mitochondriale eigenschappen van menselijke embryonale stamcellen (hESC &#39;s) en hun gedifferentieerde voorloper nageslacht. Een manier om te begrijpen en evalueren van de rol van mitochondriën in hESC functie en ontwikkelingsmogelijkheden is om rechtstreeks de activiteit van de mitochondriale ademhaling complexen. We beschrijven hier een hoge resolutie duidelijk inheemse gelelektroforese en vervolgens in de gel-activiteit visualisatie als een methode voor het analyseren van de vijf ademhalingsketen complexen van hESC &#39;s.</p>

Watch this Scientific Journal Video about Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells at JoVE.com

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

<p class="jove_content">In deze video laten wij u zien hoe de mitochondriale ademhalingsketen-complexen van menselijke embryonale stamcellen kunnen worden geanalyseerd met behulp van gel-activiteit assays.</p>

Peilen naar Mitochondriale complexe activiteit in menselijke embryonale stamcellen

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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