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. Croissance de cellules de levure <ol><li> Ensemencer les cellules de levure dans 30 ml de YPD (tableau 1) dans un petit flacon. Incuber à 30 ° C pendant la nuit avec agitation. </li><li> Diluer les cellules à une densité optique de 0,2 à 600 nm (DO 600 = 0,2) dans 50 ml de YPD et incuber à 30 ° C avec agitation par secousses. </li><li> Cultiver la culture jusqu&#39;à une densité optique de 1,0 à 600 nm (DO 600 = 1,0). </li><li> Centrifuger les cellules pendant 5 min à 2000 xg et jeter le surnageant. </li><li> Remettre en suspension le culot cellulaire dans 40 ml d&#39;eau stérile et centrifuger pendant 5 min à 2000 x g pour laver les cellules. Jeter le surnageant et stocker le culot cellulaire à -20 ° C. </li></ol> 2. Extraction des protéines <ol><li> Resuspendre les cellules dans 0,5 ml de tampon guanidine glacée (tableau 1) pour le dosage d&#39;excursion basse à l&#39;aide de Ni-NTA Superflow ou des billes de tampon A (tableau 1) pour immunoprécipitation. Garder les tubes sur la glace en tout temps. </li><li> Transfert à 2 ml tube de bouchon à vis. </li><li> Ajouter le même volume de billes de verre (Table des Matières). </li><li> Bead-a battu les cellules dans un mini cordon batteur (Table des Matières) pendant 2 min à température ambiante, puis le placer sur la glace pendant 2-3 min. Répétez cette opération trois fois. </li><li> Vortex à haute vitesse à 4 ° C pendant 30-60 min. Vérifier les cellules par visualisation au microscope à faire en sorte que les cellules sont lysées. Remarque: Les cellules lysées apparaîtront fantômes sombres et manquent une frontière ou une forme définie. De façon optimale au moins 80% des cellules doivent être lysées. </li><li> Percer un trou dans le fond du tube à l&#39;aide d&#39;une tige de poussée et le lieu dans une collection de 15 ml tube conique (bouchon à vis doit être lâche). </li><li> Centrifuger à 1000 g pendant 1 min à recueillir le lysat. </li><li> Transférer le lysat dans un tube de centrifugeuse micro. </li><li> Centrifuger à 15 000 g pendant 30 min à 4 ° C pour récupérer les protéines extraites. </li><li> Mesurer la concentration des protéines extraites à l&#39;aide de la protéine assakit y (Table des matières) et de normaliser tous les extraits à contiennent la même quantité de protéines. Porter le volume total à 1 ml avec un tampon approprié. Note: Dans la région de 5 mg de protéine totale est obtenue à partir de 50 OD 600 cellules. </li><li> Enregistrer 50 ul (250 pg de protéine si la protéine totale extraite de l&#39;étape 5 est 2,10 mg) sous forme d&#39;extrait de cellules entières (WCE). Ajouter 50 ul de tampon d&#39;échantillon Laemmli 2x (tableau 1) et incuber à 100 ° C dans un bloc chauffant à 3-5 min. Charge 10 pi de chaque échantillon dans un gel SDS-PAGE pour l&#39;analyse Western Blot (Section 5: analyse Western blot). </li></ol> 3. Purification de HF SMT3 conjugués <ol><li> Obtenir des billes de Ni-NTA Superflow (Table des Matières) nécessaires pour les expériences (100 ul de billes par exemple) par centrifugation à basse vitesse (800-1,500 xg) pendant 1 min. Retirer le surnageant. </li><li> Laver les perles 5x avec 1 ml de PBS (Table des Matières): Inverser haut-sur-bas jusqu&#39;à ce que les billes sont remises en suspension, de recueillir les perles par centrifugation à basse vitesse, et retirer le surnageant. </li><li> Suspension de billes dans 1 ml de tampon guanidine (tableau 1) et les aliquoter dans le nombre de tubes qui correspondent à des échantillons à traiter. Recueillir des perles par centrifugation à basse vitesse, et retirer le surnageant. Mélanger les perles bien en inversant haut sur le bas. Remarque: Mélanger perles bien en inversant bottom top-over. </li><li> Pour chaque échantillon, ajouter 950 ul de la WCE restant (de l&#39;étape 2.11: Extraction de protéines) à 100 ul de billes de Ni-NTA Superflow de. </li><li> Incuber sur une plate-forme à bascule à 4 ° C pendant au moins 4 heures ou toute la nuit. </li><li> Centrifugeuse à 800-1,500 g pendant 1 min à 4 ° C. </li><li> Économisez 50 pi comme surnageant (SUP). Ajouter 50 ul de tampon d&#39;échantillon Laemmli 2x et incuber à 100 ° C dans un bloc chauffant à 3-5 min. Charge 10 pi de chaque échantillon dans un gel SDS-PAGE pour l&#39;analyse Western Blot (Section 5: anal Western blotana-). </li><li> Laver les perles une fois avec 1 ml de tampon de guanidine pour 5-10 minutes sur une plate-forme à bascule. </li><li> Laver les perles 5x avec 1 ml de tampon de rupture (tableau 1) pendant 5-10 minutes sur une plate-forme à bascule. </li><li> perles de remettre en suspension dans 90 ul de tampon d&#39;échantillon Laemmli 2x. </li><li> Ajouter 10 pl de 1 M d&#39;imidazole. Note: Imidazole aide à dissocier la protéine marquée par His des Ni-NTA beads due à l&#39;interaction entre concurrence imidazole et les perles. </li><li> Incuber à 100 ° C dans un bloc chauffant pendant 3-5 min. </li><li> Vortex, puis centrifuger à 13 000 g pendant 30 sec. Transférer le surnageant dans un nouveau tube. </li><li> Charge 10-20 pi de chaque échantillon dans un gel SDS-PAGE pour l&#39;analyse Western Blot (Section 5: analyse Western blot). Remarque: ul 10-20 correspond à 0,5-1,0 mg de l&#39;entrée, si 5 mg de WCE est utilisé. </li></ol> 4. Immunoprécipitation des protéines kinétochoriens marqué par myc <ol><li> Obtenez-c-Myc lutte contre affinité gel d&#39;agarose untibody (Table des Matières) nécessaire pour les expériences (25 ul par échantillon de résine) par centrifugation à basse vitesse (800-1,500 xg). Retirer le surnageant. </li><li> Laver 5x de résine avec 1 ml de tampon A: Inverser haut-over-bas jusqu&#39;à ce que la résine est remis en suspension, recueillent résine par centrifugation à basse vitesse, et retirer le surnageant. </li><li> Suspension de résine dans 1 ml de tampon A et les aliquoter dans le nombre de tubes qui correspondent à des échantillons à traiter. Recueillir résine par centrifugation à basse vitesse, et retirer le surnageant. Remarque: Mélanger la résine et en inversant haut-over-bas. </li><li> Ajouter 950 ul de la WCE restant (de l&#39;étape 2.11: Extraction de protéines) à 25 ul de résine. </li><li> Incuber sur un agitateur oscillant à 4 ° C pendant une nuit. </li><li> Centrifugeuse à 800-1,500 g pendant 1 min à 4 ° C. </li><li> Économisez 50 pi comme surnageant (SUP). Ajouter 50 ul de tampon d&#39;échantillon Laemmli 2x et incuber à 100 ° C dans un bloc chauffant à 3-5 min. Loannonce 10 pi de chaque échantillon dans un gel SDS-PAGE pour l&#39;analyse Western Blot (Section 5: analyse Western blot). </li><li> Laver 5x de résine avec 1 ml de tampon A: Inverser haut-over-bas jusqu&#39;à ce que la résine est remis en suspension, recueillent résine par centrifugation à basse vitesse, et retirer le surnageant. </li><li> Remettre en suspension la résine dans 100 ul de tampon d&#39;échantillon de SUMEB (tableau 1). Remarque: un tampon d&#39;échantillon de SUMEB contient 8 M d&#39;urée et 1% de SDS (état dénaturant fort). </li><li> Incuber à 100 ° C dans un bloc chauffant pendant 3-5 min. </li><li> Vortex, puis centrifuger à 13 000 g pendant 30 sec. Transférer le surnageant dans un nouveau tube. </li><li> Charge 10-20 pi de chaque échantillon dans un gel SDS-PAGE pour l&#39;analyse Western Blot (Section 5: analyse Western blot). Remarque: ul 10-20 correspond à 0,5-1,0 mg de l&#39;entrée, si 5 mg de WCE est utilisé. </li></ol> 5. Analyse Western Blot <ol><li> Charger les extraits de protéines de 4-12% de gels Bis-Tris (Table des Matières) et perfélectrophorèse ORM à 120 V pendant 90 min (tampon de marche: Table des matières). </li><li> Transfert de protéines à partir de gel sur une membrane de nitrocellulose (Table des Matières) en utilisant un appareil de transfert à 30 V pendant 90 minutes (tampon de transfert: Table des Matières). </li><li> Bloquer la membrane dans 5% de lait / 1x TBST (tableau 1) pendant 1 heure à température ambiante. </li><li> Incuber la membrane avec l&#39;anticorps primaire (Table des Matières) dans 5% de lait / 1x TBST nuit à 4 ° C. Pour les anticorps primaires, en utilisant une dilution de 1: 1000 (anticorps anti-Flag et anti-Ub) ou 1: 5 000 (anticorps anti-Myc). </li><li> Laver 3x de la membrane pendant 10 min chacun avec 1x TBST. </li><li> Incuber la membrane avec l&#39;anticorps secondaire (Table des Matières) dans 5% de lait / 1x TBST pendant 1 heure à température ambiante. Utiliser une dilution 1: 5000 d&#39;anticorps secondaire. </li><li> Laver 3x de la membrane pendant 10 min chacun avec 1x TBST. </li><li> Incuber membrane avec ECL workisolution ng (Table des Matières) pendant 5 min. </li><li> Exposer la membrane sensible au bleu le film X-Ray (Table des matières) et de développer l&#39;aide d&#39;un développeur automatique (Table des matières). </li></ol>

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	<li>Johnson, P. 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.

marqueurs d&#39;épitopes tels que HA, Myc, Drapeau, et la TPS sont largement utilisés pour l&#39;analyse biochimique des protéines. Construction de souches avec HF-Smt3 et protéines kinétochoriens marqué par myc, comme Ndc10 et Ndc80, facilite la détection des PTM tels que la sumoylation et ubiquitination. HF-Smt3 déroulant test permet la détection de protéines kinétochoriens sumoylated, Ndc10 et Ndc80 (Figure 1). Le protocole de purification par affinité et analyse western blot en utilisant...

L&#39;ubiquitination et sumoylation permettent la conjugaison de l&#39;ubiquitine et le modificateur de petite ubiquitine (SUMO; Smt3 dans S. cerevisiae 1) à une protéine cible, respectivement. PTM de protéines kinétochoriens affectent leurs niveaux cellulaires et des interactions protéine-protéine au cours des différentes phases du cycle cellulaire afin d&#39;assurer la ségrégation des chromosomes fidèle. Par exemple, les niveaux cellulaires de CSE4 / CENP-A et la protéine de kinetochore extérieure DSN1 sont réglementés par protéolyse ubiquitine pour assurer la stabilité du génome 2-5. La déstabilisation de équipements kinétochoriens-microtubules incorrectes nécessite la kinase B IPL1 / Aurora, qui phosphoryle et complexes Dam1 Ndc80 qui interagissent directement avec les microtubules 8.6. Malgré l&#39;identification de plus de soixante-dix protéines kinétochoriens, il ya très peu d&#39;études qui examinent les modifications de ces protéines avec PTM, par exemple, l&#39;ubiquitine et SUMO. Une limitation importante est la capacité à préserver la PTMs pendant la purification et la rareté des anticorps sur mesure pour la détection de la sumoylation PTM tels que, la phosphorylation, la méthylation, et d&#39;autres. Caractérisation des protéines kinétochoriens sumoylated Ndc10, CEP3, BIR1 et Ndc80 utilisé un anticorps personnalisé 9. En outre, Ndc10 a été impliquée en tant que substrat pour l&#39;ubiquitination 10. HEC1 humain (Ndc80 chez S. cerevisiae) est également substrat pour ubiquitination, réglementé par APC / C-hCdh1 ligase E3 11. Par conséquent, Ndc10 et Ndc80 sont de bons candidats pour l&#39;optimisation du protocole de détecter à la fois la sumoylation et ubiquitination en S. cerevisiae. Pour faciliter l&#39;identification de la sumoylation, nous avons construit des souches qui expriment HF-Smt3 et Myc-tagged Ndc10 ou Ndc80. L&#39;utilisation de marqueurs d&#39;epitope (HF: His6-Flag) minimise le bruit de fond dû à la réactivité croisée qui est fréquemment observé dans le sérum polyclonal dirigé contre une protéine candidate. Nous avons élaboré un protocole d&#39;affinitépurifier les conjugués HF-SMT3 commercial et ensuite utilisé des anticorps anti-Myc et anti-Flag pour détecter la présence de sumolyated Ndc10 et Ndc80 dans la préparation purifiée Smt3. Pour ubiquitination, nous avons conçu un protocole d&#39;immunoprécipitation modifiée qui conserve ubiquitination des protéines kinétochoriens marqué par myc et effectué une analyse western blot avec un anticorps commercial anti-Ub pour détecter ubiquitination de Ndc10 et 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

Les modifications post-traductionnelles (PTM), tels que la phosphorylation, la méthylation, l&#39;acétylation, l&#39;ubiquitination, et sumoylation, régulent la fonction cellulaire de nombreuses protéines. PTM de protéines kinétochoriens qui associent avec l&#39;ADN centromérique médiation ségrégation des chromosomes fidèle à maintenir la stabilité du génome. Approches biochimiques telles que la spectrométrie de masse et analyse western blot sont les plus couramment utilisés pour l&#39;identification des PTM. Ici, un procédé de purification des protéines est décrite qui permet la détection à la fois de la sumoylation et ubiquitination des protéines kinétochoriens, Ndc10 et Ndc80, dans Saccharomyces cerevisiae. Une souche qui exprime polyhistidine-marquage Flag Smt3 (HF-Smt3) et marqué par myc Ndc10 ou Ndc80 a été construit et utilisé pour nos études. Pour la détection de la sumoylation, nous avons conçu un protocole pour purifier par affinité les protéines His-étiqueté sumoylated à l&#39;aide de billes de nickel et utilisé une analyse western blot avec un anticorps anti-Myc pour détecter une sumoylated Ndc10e Ndc80. Pour la détection de l&#39;ubiquitination, nous avons élaboré un protocole pour l&#39;immunoprécipitation des protéines Myc-marqués et utilisé l&#39;analyse de western blot avec l&#39;anticorps anti-Ub pour montrer que Ndc10 et Ndc80 sont ubiquitinées. Nos résultats montrent que l&#39;épitope protéine marquée de l&#39;intérêt pour l&#39;His-Drapeau étiqueté souche Smt3 facilite la détection de plusieurs PTM. Les études futures devraient permettre une exploitation de cette technique pour identifier et caractériser les interactions entre protéines qui sont tributaires d&#39;un PTM spécifique.

Pour détecter sumoylation de protéines kinétochoriens Ndc80 et Ndc10, les souches avec HF-Smt3 et protéines kinétochoriens marqué par myc (Ndc80 ou Ndc10) ont été construits (tableau 2), comme décrit précédemment 9,12. Conjugués HF-SMT3 ont été purifiés par affinité en utilisant des billes de Ni-NTA. Analyse par Western blot purifié HF-Smt3 avec un anticorps anti-Flag a permis la détection de formes sumoylated de substrats SUMO qui étaient absentes dans la souche de contrô...

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

Ce manuscrit décrit la détection de la sumoylation de protéines et ubiquitination kinétochoriens, Ndc10 Ndc80 et, dans le bourgeonnement levure Saccharomyces cerevisiae.

Protein Purification Technique qui permet la détection de sumoylation et Ubiquitination de levure bourgeonnante kinétochoriens protéines Ndc10 et Ndc80

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

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Biology

12.0K vues.  National Cancer Institute, National Institute of Health.  Ce manuscrit décrit la détection de la sumoylation de protéines et ubiquitination kinétochoriens, Ndc10 Ndc80 et, dans le bourgeonnement levure Saccharomyces cerevisiae. 

Vidéo: 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.

Mesure Life Span réplicative dans la levure bourgeonnante

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

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Biologie

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.

La détection des protéines ubiquitination

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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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.

Purification des mitochondries de cellules de levure

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.

Analyse du développement d&#39;un phénotype morphologique en fonction de la concentration de protéines dans la levure bourgeonnante

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.

Purification de Hsp104, une protéine 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.

Purification chromatographique des ribosomes de levure hautement 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;acquisition de fluorescence Time-lapse films de levure bourgeonnante et analyse de la dynamique de cellules uniques en utilisant des greffes

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.

Bourgeonnement de levure extraction et purification des protéines pour l&#39;étude de la fonction, Interactions, et des modifications post-traductionnelles

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 Expression dépistage des protéines membranaires dans<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 ...