Name: in situ subcellular fractionation of adherent and non-adherent mammalian cells
Uploaded: 2010-07-23T17:30:00
Description: Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

Anyaporn Sawasdichai en Nazefah Abdul Hamid Met dank aan de Koninklijke Thaise regering en de regering van Maleisië respectievelijk Ph.D. Beurzen. Dit werk werd gefinancierd door een Wellcome Trust projectsubsidie ​​toegekend aan PSJ en KG. We zijn ook dankbaar voor de Universiteit van Bristol Wolfson BioImaging Facility.

I. Voorbereiding voor fractionering Dit gedeelte beschrijft de bereiding van poly-L-lysine gecoate dekglaasjes microscoop en de aanhechting van cellen voorafgaand aan de fractionering. Indien gewenst kunnen de cellen tijdelijk getransfecteerd worden met eiwit expressievectoren voor of na bevestiging. A. Bereiding van poly-L-Lysine beklede dekglaasjes <ol><li> Bereid een oplossing van 1mg/ml poly-L-lysine in gedistilleerd water. </li><li> Vacht schoon coverslips m...

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	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

Vaak voorkomende problemen en suggesties: Alle of de meeste van de cellen verloren zijn gegaan tijdens het wassen. Tijdens het wassen stappen vloeistoffen moeten langzaam worden toegevoegd aan de kant van de 6-wells plaat het vermijden van de dekglaasje aan. Op dezelfde vloeistoffen moeten worden verwijderd door voorzichtig kantelen van de plaat en langzaam pipetteren overtollige vloeistof. Celadhesie kan worden verhoogd met behulp van poly-L-Lysine beklede dekglaasjes. <p class="jove_conte...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
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in situ subcellular fractionation of adherent and non-adherent mammalian cells

Eiwit-functie is nauw gekoppeld aan eiwit lokalisatie. Hoewel sommige eiwitten zijn beperkt tot een specifieke locatie of subcellulaire compartiment, veel eiwitten aanwezig zijn als een vrij verspreiden van de bevolking in de vrije uitwisseling met een sub-populatie die nauw is gekoppeld aan een bepaalde subcellulaire domein of structuur. In situ subcellulaire fractionering kan de visualisatie van eiwit verkokering en kan ook zien eiwit subpopulaties die lokaliseren om specifieke structuren. Bijvoorbeeld, kan verwijdering van de oplosbare cytoplasmatische eiwitten en losjes nucleaire eiwitten onthullen de stabiele vereniging van een aantal transcriptiefactoren met chromatine. Latere spijsvertering van DNA kan in sommige gevallen openbaren associatie met het netwerk van eiwitten en RNA&#39;s die collectief wordt genoemd de nucleaire steiger of nucleaire matrix. Hier beschrijven we de stappen die nodig zijn tijdens de in situ fractionering van het aanklevende en niet-hechtende cellen van zoogdieren op microscoop dekglaasjes. Eiwit visualisatie kan worden bereikt met behulp van specifieke antilichamen of TL-fusie-eiwitten en fluorescentie microscopie. Antilichamen en / of fluorescente kleurstoffen die fungeren als markers voor specifieke vakken of structuren laten eiwit lokalisatie in kaart worden gebracht in detail. In situ fractionering kan ook worden gecombineerd met western blotting om de hoeveelheden eiwit aanwezig is in elke fractie vergelijken. Deze eenvoudige biochemische benadering kan onthullen verenigingen die anders onopgemerkt zouden blijven.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellulaire fractionatie van zoogdiercellen op microscoop dekglaasjes kan de visualisatie van eiwit lokalisatie.

In situ subcellulaire fractionering van het aanklevende en niet-klevende zoogdiercellen

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

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Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

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RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

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Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...