We thank Dr. Oliver Kerscher for support and advice and members of the Basrai laboratory for their support and comments on the paper. This work was supported by the National Institutes of Health Intramural Research Program.

1. La crescita di cellule di lievito <ol><li> Inoculare cellule di lievito in 30 ml di YPD (Tabella 1) in un palloncino. Incubare a 30 ° C per una notte con agitazione. </li><li> Diluire le cellule ad una densità ottica di 0,2 a 600 nm (OD 600 = 0.2) in 50 ml di YPD e incubare a 30 ° C con agitazione. </li><li> Crescere la coltura ad una densità ottica di 1,0 a 600 nm (OD 600 = 1.0). </li><li> Celle di centrifugazione per 5 min a 2000 xg e scartare il surnatante. </li><li> Risospendere il pellet di cellule in 40 ml di acqua sterile e centrifugare per 5 min a 2000 xg per lavare le cellule. Eliminare il surnatante e conservare il pellet cellulare a -20 ° C. </li></ol> 2. Estrazione di proteine <ol><li> Risospendere le cellule in 0,5 ml di ghiacciata buffer di guanidina (Tabella 1) per il dosaggio di pull-down con Ni-NTA perline Superflow o tampone A (Tabella 1) per immunoprecipitazione. Tenere tubi sul ghiaccio in ogni momento. </li><li> Trasferimento a 2 ml tubo tappo a vite. </li><li> Aggiungere lo stesso volume di perline di vetro (Table of Materials). </li><li> Bead-battere le celle di una mini tallone battitore (tabella dei materiali) per 2 minuti a temperatura ambiente, poi posto in ghiaccio per 2-3 minuti. Ripetere questa operazione tre volte. </li><li> Vortex sulla alta velocità a 4 ° C per 30-60 min. Controllare le cellule mediante visualizzazione al microscopio per assicurare che le cellule sono lisate. Nota: cellule lisate appariranno fantasmi scuri e la mancanza di un confine o una forma definita. In modo ottimale almeno l&#39;80% delle cellule dovrebbe essere lisato. </li><li> Perforare un foro nella parte inferiore del tubo con una puntina e posto in una collezione tubo da 15 ml (tappo a vite deve essere sciolto). </li><li> Centrifugare a 1000 xg per 1 minuto per raccogliere il lisato. </li><li> Trasferire il lisato in una provetta da centrifuga micro. </li><li> Centrifugare a 15.000 xg per 30 min a 4 ° C per raccogliere le proteine ​​estratte. </li><li> Misurare la concentrazione delle proteine ​​estratte usando l&#39;assa proteiney Kit (Tabella dei Materiali) e normalizzare tutti gli estratti per contenere la stessa quantità di proteine. Portare il volume totale di 1 ml con tampone appropriato. Nota: Circa 5 mg di proteine ​​totali è ottenuta da 50 OD 600 cellule. </li><li> Risparmia il 50 microlitri (250 ug di proteine ​​se la proteina totale estratto dal punto 2.10 è di 5 mg), come estratto di cellula intera (WCE). Aggiungere 50 ml di tampone campione 2x Laemmli (Tabella 1) e incubare a 100 ° C in un blocco di calore per 3-5 min. Carico 10 ml di ogni campione in un gel SDS-PAGE per Western Blot analisi (Sezione 5: analisi Western Blot). </li></ol> 3. Purificazione di HF-Smt3 Coniugati <ol><li> Ottenere le perline Superflow Ni-NTA (Tabella dei Materiali) necessari per gli esperimenti (100 ml di perline per campione) tramite centrifugazione a bassa velocità (800-1,500 xg) per 1 min. Rimuovere il surnatante. </li><li> Lavare perline 5x con 1 ml di PBS (Table of Materials): Inverti top-overfondo fino a quando le perle sono risospese, raccogliere perle di centrifugazione a bassa velocità, e rimuovere il surnatante. </li><li> Sospendere perline in 1 ml di tampone guanidina (Tabella 1) e un&#39;aliquota nel numero di tubi corrispondenti ai campioni da trattare. Raccogliere perle per centrifugazione a bassa velocità, e rimuovere il surnatante. Mescolare le perline ben invertendo top-over in basso. Nota: Miscelare cordoni ben invertendo bottom top-over. </li><li> Per ciascun campione, aggiungere 950 microlitri della restante WCE (dal punto 2.11: Estrazione di proteine) a 100 ml di Ni-NTA perline Superflow. </li><li> Incubare su una piattaforma oscillante a 4 ° C per almeno 4 ore o tutta la notte. </li><li> Centrifugare a 800-1,500 xg per 1 min a 4 ° C. </li><li> Risparmia il 50 microlitri come surnatante (SUP). Aggiungere 50 ml di tampone campione 2x Laemmli e incubare a 100 ° C in un blocco di calore per 3-5 min. Carico 10 ml di ogni campione in un gel SDS-PAGE per Western Blot analisi (Sezione 5: anal Western BlotYsis). </li><li> Perline Lavare una volta con 1 ml di tampone guanidina per 5-10 minuti su una piattaforma oscillante. </li><li> Lavare perline 5x con 1 ml di tampone di rottura (Tabella 1) per 5-10 minuti su una piattaforma oscillante. </li><li> Perline Risospendere in 90 ml di tampone campione 2x Laemmli. </li><li> Aggiungere 10 ml di 1 M imidazolo. Nota: Imidazolo aiuta a dissociarsi proteina His-tag da perline Ni-NTA dovuta all&#39;interazione competizione tra imidazolo e le perle. </li><li> Incubare a 100 ° C in un blocco di calore per 3-5 min. </li><li> Vortex, quindi centrifugare a 13.000 g per 30 sec. Trasferire il surnatante in una nuova provetta. </li><li> Carico 10-20 ml di ogni campione in un gel SDS-PAGE per Western Blot analisi (Sezione 5: analisi Western Blot). Nota: 10-20 ml corrisponde a 0,5-1,0 mg di ingresso, se viene utilizzato 5 mg di WCE. </li></ol> 4. Immunoprecipitation di Myc-tag cinetocore Proteine <ol><li> Ottenere anti-c-Myc affinità agarosio gel untibody (Tabella dei Materiali) necessari per gli esperimenti (25 microlitri di resina per campione) tramite centrifugazione a bassa velocità (800-1,500 xg). Rimuovere il surnatante. </li><li> 5x resina Lavare con 1 ml di tampone A: Inverti top-over-basso fino la resina viene risospeso, raccolgono resina tramite centrifugazione a bassa velocità, e rimuovere il surnatante. </li><li> Sospendere resina in 1 ml di tampone A e Aliquotare nel numero di tubi corrispondenti ai campioni da trattare. Raccogliere resina mediante centrifugazione a bassa velocità, e rimuovere il surnatante. Nota: Mescolare la resina ben invertendo top-over-bottom. </li><li> Aggiungere 950 microlitri della restante WCE (dal punto 2.11: Estrazione di proteine) a 25 ml di resina. </li><li> Incubare su una piattaforma oscillante a 4 ° C durante la notte. </li><li> Centrifugare a 800-1,500 xg per 1 min a 4 ° C. </li><li> Risparmia il 50 microlitri come surnatante (SUP). Aggiungere 50 ml di tampone campione 2x Laemmli e incubare a 100 ° C in un blocco di calore per 3-5 min. Load 10 ml di ogni campione in un gel SDS-PAGE per l&#39;analisi Western Blot (Sezione 5: analisi Western Blot). </li><li> 5x resina Lavare con 1 ml di tampone A: Inverti top-over-basso fino la resina viene risospeso, raccolgono resina tramite centrifugazione a bassa velocità, e rimuovere il surnatante. </li><li> Risospendere la resina in 100 ml di tampone campione SUMEB (Tabella 1). Nota: tampone campione SUMEB contiene 8 M Urea e 1% SDS (forte condizione di denaturazione). </li><li> Incubare a 100 ° C in un blocco di calore per 3-5 min. </li><li> Vortex, quindi centrifugare a 13.000 g per 30 sec. Trasferire il surnatante in una nuova provetta. </li><li> Carico 10-20 ml di ogni campione in un gel SDS-PAGE per Western Blot analisi (Sezione 5: analisi Western Blot). Nota: 10-20 ml corrisponde a 0,5-1,0 mg di ingresso, se viene utilizzato 5 mg di WCE. </li></ol> 5. Western Blot analisi <ol><li> Caricare i estratti proteici di 4-12% Bis-Tris gels (Tabella dei Materiali) e perfelettroforesi orm a 120 V per 90 min (buffer di Esecuzione: Tabella dei Materiali). </li><li> Trasferire le proteine ​​da gel su una membrana di nitrocellulosa (Table of Materials) usando un dispositivo di trasferimento a 30 V per 90 min (Buffer di trasferimento: Table of Materials). </li><li> Bloccare la membrana in 5% di latte / 1x TBST (Tabella 1) per 1 ora a temperatura ambiente. </li><li> Incubare a membrana con anticorpo primario (Table of Materials) a 5% di latte / 1x TBST notte a 4 ° C. Per anticorpi primari, utilizzare una diluizione di 1: 1.000 (anticorpi anti-Flag e anti-Ub) o 1: 5.000 (anticorpo anti-Myc). </li><li> Lavare membrana 3x per 10 minuti ciascuno con 1x TBST. </li><li> Incubare a membrana con anticorpo secondario (Tabella dei Materiali) a 5% di latte / 1x TBST per 1 ora a temperatura ambiente. Usare una diluizione 1: 5.000 del anticorpi secondari. </li><li> Lavare membrana 3x per 10 minuti ciascuno con 1x TBST. </li><li> Incubare a membrana con ECL workisoluzione ng (Table of Materials) per 5 min. </li><li> Esporre la membrana blu X-ray film sensibile (Tabella dei Materiali) e sviluppare utilizzando uno sviluppatore automatica (Table of Materials). </li></ol>

<ol>
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R., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-1:+Ubiquitin+gains+weight.">SUMO-1: Ubiquitin gains weight.</a> Trends Cell Biol. 7, 408-413 (1997).</li><li>Hewawasam, G., 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=Psh1+is+an+E3+ubiquitin+ligase+that+targets+the+centromeric+histone+variant+Cse4.">Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4.</a> Mol Cell. 40, 444-454 (2010).</li><li>Ranjitkar, P., 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=An+E3+ubiquitin+ligase+prevents+ectopic+localization+of+the+centromeric+histone+H3+variant+via+the+centromere+targeting+domain.">An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain.</a> Mol Cell. 40, 455-464 (2010).</li><li>Au, W. C., 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+novel+role+of+the+N+terminus+of+budding+yeast+histone+H3+variant+Cse4+in+ubiquitin-mediated+proteolysis.">A novel role of the N terminus of budding yeast histone H3 variant Cse4 in ubiquitin-mediated proteolysis.</a> Genetics. 194, 513-518 (2013).</li><li>Akiyoshi, B., 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=The+Mub1/Ubr2+ubiquitin+ligase+complex+regulates+the+conserved+Dsn1+kinetochore+protein.">The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein.</a> PLoS Genet. 9, e1003216 (2013).</li><li>Biggins, 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=The+conserved+protein+kinase+Ipl1+regulates+microtubule+binding+to+kinetochores+in+budding+yeast.">The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast.</a> Genes Dev. 13, 532-544 (1999).</li><li>Cheeseman, I. M., 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=Phospho-regulation+of+kinetochore-microtubule+attachments+by+the+Aurora+kinase+Ipl1p.">Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p.</a> Cell. 111, 163-172 (2002).</li><li>Liu, D., Lampson, M. A. <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+kinetochore-microtubule+attachments+by+Aurora+B+kinase.">Regulation of kinetochore-microtubule attachments by Aurora B kinase.</a> Biochem Soc Trans. 37, 976-980 (2009).</li><li>Montpetit, B., Hazbun, T. R., Fields, S., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sumoylation+of+the+budding+yeast+kinetochore+protein+Ndc10+is+required+for+Ndc10+spindle+localization+and+regulation+of+anaphase+spindle+elongation.">Sumoylation of the budding yeast kinetochore protein Ndc10 is required for Ndc10 spindle localization and regulation of anaphase spindle elongation.</a> J Cell Biol. 174, 653-663 (2006).</li><li>Furth, N., 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=Exposure+of+bipartite+hydrophobic+signal+triggers+nuclear+quality+control+of+Ndc10+at+the+endoplasmic+reticulum/nuclear+envelope.">Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.</a> Mol Biol Cell. 22, 4726-4739 (2011).</li><li>Li, 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=Anaphase-promoting+complex/cyclosome+controls+HEC1+stability.">Anaphase-promoting complex/cyclosome controls HEC1 stability.</a> Cell Prolif. 44, 1-9 (2011).</li><li>Takahashi, Y., Yong-Gonzalez, V., Kikuchi, Y., Strunnikov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIZ1/SIZ2+control+of+chromosome+transmission+fidelity+is+mediated+by+the+sumoylation+of+topoisomerase+II.">SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II.</a> Genetics. 172, 783-794 (2006).</li><li>Liu, C., Apodaca, J., Davis, L. E., Rao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+inhibition+in+wild-type+yeast+Saccharomyces+cerevisiae+cells.">Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells.</a> Biotechniques. 42, 158 (2007).</li><li>Kitamura, E., Tanaka, K., Kitamura, Y., Tanaka, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetochore+microtubule+interaction+during+S+phase+in+Saccharomyces+cerevisiae.">Kinetochore microtubule interaction during S phase in Saccharomyces cerevisiae.</a> Genes Dev. 21, 3319-3330 (2007).</li><li>Gascoigne, K. E., Cheeseman, 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=Kinetochore+assembly:+if+you+build+it,+they+will+come.">Kinetochore assembly: if you build it, they will come.</a> Curr Opin Cell Biol. 23, 102-108 (2011).</li></ol>

The authors have nothing to disclose.

Tag epitopo quali HA, Myc, Bandiera, e GST sono ampiamente utilizzati per l&#39;analisi biochimica delle proteine. Costruzione di ceppi con HF-Smt3 e proteine ​​cinetocore Myc-tag, come Ndc10 e Ndc80, facilita l&#39;individuazione di PTM, come sumoilazione e ubiquitinazione. HF-Smt3 tirare giù test permette la rilevazione di proteine ​​cinetocore sumoylated, Ndc10 e Ndc80 (Figura 1). Il protocollo di purificazione per affinità e western blot utilizzando un anticorpo anti-Flag stabilire la spec...

Ubiquitinazione e sumoilazione permettono la coniugazione di ubiquitina e Modifier Piccolo ubiquitina-simile (SUMO, Smt3 in S. cerevisiae 1) ad una proteina bersaglio, rispettivamente. PTM di proteine ​​cinetocore influenzano i livelli cellulari e interazioni proteina-proteina durante le diverse fasi del ciclo cellulare per garantire fedele segregazione dei cromosomi. Ad esempio, i livelli cellulari di Cse4 / CENP-A e proteine ​​cinetocore esterno DSN1 sono regolate da proteolisi ubiquitina-mediata per garantire la stabilità del genoma 2-5. Destabilizzazione degli allegati cinetocore-microtubuli non corretti richiede la B chinasi Ipl1 / Aurora, che fosforila Dam1 e Ndc80 complessi che interagiscono direttamente con i microtubuli 6-8. Nonostante l&#39;identificazione di oltre settanta proteine ​​cinetocore, ci sono molto pochi gli studi che indagano le modifiche di queste proteine ​​con PTM, ad esempio, l&#39;ubiquitina e SUMO. Una limitazione importante è la capacità di conservare la PTMs durante la purificazione e la scarsità di anticorpi personalizzati per la rilevazione di PTM come sumoylation, fosforilazione, metilazione, e altri. Caratterizzazione di proteine ​​cinetocore sumoylated Ndc10, Cep3, Bir1, e Ndc80 utilizzato un anticorpo personalizzato 9. Inoltre, Ndc10 è stata implicata come substrato per 10 ubiquitinazione. Hec1 umana (Ndc80 in S. cerevisiae) è anche substrato per ubiquitinazione, regolato da APC / C-hCdh1 E3 ligasi 11. Pertanto, Ndc10 e Ndc80 sono buoni candidati per l&#39;ottimizzazione del protocollo per rilevare sia sumoilazione e ubiquitinazione in S. cerevisiae. Per facilitare l&#39;individuazione di sumoilazione, abbiamo costruito i ceppi che esprimono HF-Smt3 e Myc-tag Ndc10 o Ndc80. L&#39;uso di tag epitopi (HF: His6-Flag) riduce al minimo il fondo a causa della cross-reattività che è frequente nel siero policlonale sollevata contro una proteina candidato. Abbiamo ideato un protocollo per affinitàpurificare coniugati HF-Smt3 e poi utilizzati commerciale anti-Flag e anticorpi anti-myc per rilevare la presenza di sumolyated Ndc10 e Ndc80 nella preparazione Smt3 purificato. Per ubiquitination, abbiamo ideato un protocollo immunoprecipitazione modificata che conserva ubiquitinazione delle proteine ​​cinetocore Myc-targhette e svolta western blot con l&#39;anticorpo commerciale anti-Ub per rilevare ubiquitinazione di Ndc10 e Ndc80.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass beads</td>
			<td>BioSpec Products</td>
			<td align="right">11079105</td>
			<td>0.5 mm dia.</td>
		</tr>
		<tr>
			<td>Mini beadbeater</td>
			<td>BioSpec Products</td>
			<td>#693</td>
			<td>8 cell disrupter</td>
		</tr>
		<tr>
			<td>Ni-NTA superflow</td>
			<td>Qiagen</td>
			<td align="right">30430</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8215</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Anti-c-Myc agarose affinity gel antbody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>A7470</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Monoclonal anti-Flag M2 antibody produced in mouse</td>
			<td>Sigma-Aldrich</td>
			<td>F1804</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>c-Myc antibody (A-14)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-789</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Purified mouse antibody monoclonal 9E10</td>
			<td>Covance</td>
			<td>MMS-150P</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Ubiquitin (P4G7) monoclonal antibody</td>
			<td>Covance</td>
			<td>MMS-258R</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>ECL Rabbit IgG, HRP-linked whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA934V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>ECL Mouse IgG, HRP-Linked Whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA931V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>DC protein assay</td>
			<td>Bio-Rad</td>
			<td>500-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Novex</td>
			<td>LC2001</td>
			<td>0.45 mm pore size</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Novex</td>
			<td>NP0321BOX</td>
			<td>1.0 mm, 10 well</td>
		</tr>
		<tr>
			<td>NuPAGE MES SDS Running Buffer</td>
			<td>Novex</td>
			<td>NP0002</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Novex</td>
			<td>NP0006-1</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td align="right">34078</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS pH7.4</td>
			<td>GIBCO</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue sensitive X-Ray film</td>
			<td>Dbio</td>
			<td>DBOF30003</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic developer</td>
			<td>Kodak</td>
			<td>M35AX-OMAT</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>MG-132</td>
			<td>Selleck Chemicals</td>
			<td>S2619</td>
			<td>25 mg</td>
		</tr>
	</tbody>
</table>

protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80

Modificazioni post-traduzionali (PTM), come fosforilazione, metilazione, acetilazione, ubiquitinazione e sumoilazione, regolano la funzione cellulare di molte proteine. PTM di proteine ​​cinetocore che associano con il DNA centromerico mediano fedele segregazione dei cromosomi per mantenere la stabilità del genoma. Approcci biochimici come la spettrometria di massa e analisi di western blot sono più comunemente utilizzati per l&#39;identificazione di PTM. Qui, un metodo di purificazione della proteina è descritto che permette il rilevamento di entrambe sumoylation e ubiquitinazione delle proteine ​​cinetocore, Ndc10 e Ndc80, in Saccharomyces cerevisiae. Un ceppo che esprime polyhistidine-Flag-tagged Smt3 (HF-Smt3) e Myc-tagged Ndc10 o Ndc80 è stato costruito e utilizzato per i nostri studi. Per il rilevamento di sumoylation, abbiamo ideato un protocollo per affinità purificare proteine ​​sumoylated His-tagged utilizzando perline di nichel e usato western blot con anticorpi anti-Myc per rilevare sumoylated Ndc10 unnd Ndc80. Per il rilevamento di ubiquitinazione, abbiamo ideato un protocollo per immunoprecipitazione di proteine ​​Myc-tag e utilizzato analisi Western Blot con anticorpi anti-Ub per dimostrare che Ndc10 e Ndc80 sono ubiquitinate. I nostri risultati mostrano che epitopi di proteine ​​tag di interesse per il suo-Flag tagged ceppo Smt3 facilita l&#39;individuazione di molteplici PTM. Studi futuri dovrebbero consentire lo sfruttamento di questa tecnica per identificare e caratterizzare le interazioni proteina che dipendono da uno specifico PTM.

Per rilevare sumoylation di proteine ​​cinetocore Ndc80 e Ndc10, ceppi con HF-Smt3 e proteine ​​cinetocore Myc-tag (Ndc80 o Ndc10) sono stati costruiti (Tabella 2), come descritto in precedenza 9,12. Coniugati HF-Smt3 stati affinità purificate utilizzando Ni-NTA perline. Analisi Western blot di purificato HF-Smt3 con un anticorpo anti-Flag permesso rilevamento di forme sumoylated di substrati SUMO che erano assenti nel ceppo di controllo senza HF-Smt3 (Figura 1A e <s...

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Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Questo manoscritto descrive la rilevazione di sumoilazione e ubiquitinazione delle proteine ​​cinetocore, Ndc10 e Ndc80, nel lievito Saccharomyces cerevisiae erba.

Purificazione delle proteine ​​tecnica che permette la rilevazione di sumoilazione e Ubiquitinazione di lievito in erba cinetocore proteine ​​Ndc10 e Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

protein-purification-technique-that-allows-detection-sumoylation

Research

JoVE Journal

Biology

12.0K Views.  National Cancer Institute, National Institute of Health.  Questo manoscritto descrive la rilevazione di sumoilazione e ubiquitinazione delle proteine ​​cinetocore, Ndc10 e Ndc80, nel lievito Saccharomyces cerevisiae erba. 

Video: Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Durata pannello misura replicazione in lievito

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Ricerca

Biologia

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Rilevamento di proteine ​​Ubiquitinazione

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificazione dei mitocondri da cellule di lievito

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Analisi dello sviluppo di un fenotipo morfologico come funzione della concentrazione di proteine ​​in lievito

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purificazione di Hsp104, una proteina Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificazione cromatografica dei ribosomi di lievito Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

L&#39;acquisizione di fluorescenza filmati time-lapse di lievito in erba e Analisi Dinamica singola cella utilizzando innesti

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Purificazione delle proteine ​​tecnica che permette la rilevazione di sumoilazione e Ubiquitinazione di lievito in erba cinetocore proteine ​​Ndc10 e Ndc80

Purificazione delle proteine tecnica che permette la rilevazione di sumoilazione e Ubiquitinazione di lievito in erba cinetocore proteine Ndc10 e Ndc80