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. Wachstum von Hefezellen, <ol><li> Inokulieren Hefezellen in 30 ml YPD (Tabelle 1) in einem kleinen Kolben. Inkubation bei 30 ° C über Nacht unter Schütteln. </li><li> Verdünne die Zellen bis zu einer optischen Dichte von 0,2 bei 600 nm (OD 600 = 0,2) in 50 ml YPD und Inkubieren bei 30 ° C unter Schütteln inkubiert. </li><li> Die Kultur auf eine optische Dichte von 1,0 bei 600 nm (OD 600 = 1,0). </li><li> Centrifuge Zellen für 5 min bei 2.000 xg und den Überstand verwerfen. </li><li> Zellpellet in 40 ml sterilem Wasser und Zentrifuge für 5 min bei 2.000 xg, um die Zellen zu waschen. Überstand verwerfen und speichern Sie das Zellpellet bei -20 ° C. </li></ol> 2. Extraktion von Proteinen <ol><li> Die Zellen in 0,5 ml eiskaltem Guanidinpuffer (Tabelle 1) für die Pull-Down-Assay unter Verwendung von Ni-NTA-Superflow Perlen oder Puffer A (Tabelle 1) für die Immunfällung. Halten Röhrchen auf Eis zu allen Zeiten. </li><li> Transfer zu 2 ml Schraubverschluss Röhrchen. </li><li> In die gleiche Menge Glasperlen (Table of Materials). </li><li> Bead-schlagen, die Zellen in einem Mini Bead Beater (Table of Materials) für 2 Minuten bei Raumtemperatur, dann auf Eis legen für 2-3 min. Wiederholen Sie diesen Vorgang dreimal. </li><li> Wirbel mit hoher Geschwindigkeit bei 4 ° C für 30-60 min. Überprüfen Sie die Zellen durch Visualisierung unter dem Mikroskop, um sicherzustellen, daß die Zellen lysiert. Hinweis: lysierten Zellen werden als dunkle Geister erscheinen und es fehlt ihnen eine Grenze oder definierte Form. In optimaler Weise mindestens 80% der Zellen lysiert werden sollte. </li><li> Punktion ein Loch in den Boden des Röhrchens mit einem Push-Pin und Ort in einer Sammlung enthalten 15 ml konischen Rohr (Schraubdeckel sollte locker sein). </li><li> Zentrifuge bei 1000 × g für 1 min, um das Lysat zu sammeln. </li><li> Transfer des Lysats auf einem Mikrozentrifugenröhrchen. </li><li> Zentrifuge bei 15.000 g für 30 min bei 4 ° C, um die extrahierten Proteine ​​zu sammeln. </li><li> Messung der Konzentration der extrahierten Proteine ​​mit dem Protein assay Kit (Table of Materials) und normalisieren extrakte, um die gleiche Menge an Protein enthalten. Bringen des Gesamtvolumens auf 1 ml mit einem geeigneten Puffer. Anmerkung: Etwa 5 mg Gesamtprotein aus 50 OD 600-Zellen erhalten. </li><li> Sparen Sie 50 ul (250 ug Protein, wenn das Gesamtprotein aus Schritt 2.10 extrahiert ist 5 mg) als Gesamtzellextrakt (WCE). Dann werden 50 ul 2x Laemmli Probenpuffer (Tabelle 1) und Inkubation bei 100 ° C in einem Heizblock für 3-5 min. Last 10 ul jeder Probe in einem SDS-PAGE-Gel für Western Blot-Analyse (Abschnitt 5: Western-Blot-Analyse). </li></ol> 3. Reinigung von HF-Smt3 Konjugate <ol><li> Besorgen Sie sich die Ni-NTA-Superflow-Perlen (Table of Materials) für die Versuche (100 &amp; mgr; l Kügelchen pro Probe) durch Zentrifugation bei niedriger Geschwindigkeit (800-1500 xg) für 1 min notwendig. Entfernen Sie den Überstand. </li><li> Wash Perlen 5x mit 1 ml PBS (Table of Materials): Invert top-Über-unten, bis die Kügelchen wieder suspendiert werden, sammeln Perlen durch Zentrifugation bei niedriger Geschwindigkeit, und entfernen Sie den Überstand. </li><li> Aussetzen Kügelchen in 1 ml Guanidin-Puffer (Tabelle 1) und aliquotiert sie in der Anzahl der Rohre entsprechenden Proben zu verarbeiten. Sammle Perlen durch Zentrifugation bei niedriger Geschwindigkeit, und entfernen Sie den Überstand. Mischen Sie die Perlen auch durch Umdrehen über top-bottom. Hinweis: Mischen Sie Perlen gut durch Umkehren oben über Boden. </li><li> Für jede Probe wird mit 950 &amp; mgr; l der verbleibenden WCE (aus Schritt 2.11: Extraktion der Proteine) zu 100 ul Ni-NTA Superflow Beads. </li><li> Inkubieren auf einer Schüttelplattform bei 4 ° C für mindestens 4 Stunden oder über Nacht. </li><li> Zentrifuge bei 800-1500 x g für 1 Minute bei 4 ° C. </li><li> Sparen Sie 50 ul als Überstand (SUP). Dann werden 50 ul 2x Laemmli Probenpuffer und Inkubation bei 100 ° C in einem Heizblock für 3-5 min. Last 10 ul jeder Probe in einem SDS-PAGE-Gel für Western Blot-Analyse (Abschnitt 5: Western-Blot-anallyse). </li><li> Wasch Kügelchen einmal mit 1 ml Guanidin-Puffer für 5-10 min auf einer Schwingplattform. </li><li> Wash Perlen 5x mit 1 ml Aufbruchpuffer (Tabelle 1) für 5-10 min auf einem Wippschüttler. </li><li> Resuspendieren Perlen in 90 ul 2x Laemmli-Probenpuffer. </li><li> Zugabe von 10 ul 1 M Imidazol. Hinweis: Imidazol hilft, die His-Tag-Protein aus der Ni-NTA-Perlen aufgrund konkurrierender Wechselwirkungen zwischen Imidazol und die Perlen zu distanzieren. </li><li> Inkubation bei 100 ° C in einem Heizblock für 3-5 min. </li><li> Vortex, dann bei 13.000 xg zentrifugieren für 30 Sekunden. Den Überstand in ein frisches Röhrchen. </li><li> Last 10-20 ul jeder Probe in einem SDS-PAGE-Gel für Western Blot-Analyse (Abschnitt 5: Western-Blot-Analyse). Anmerkung: 10-20 ul entspricht 0,5-1,0 mg Eingang, wenn 5 mg WCE verwendet. </li></ol> 4. Immunpräzipitation von Myc-markierten Proteine ​​Kinetochor <ol><li> Erhalten anti-c-Myc Agarose Affinitätsgel eintibody (Table of Materials) für die Versuche (25 ul Harz pro Probe) durch Zentrifugation bei niedriger Geschwindigkeit (800-1500 xg) notwendig. Entfernen Sie den Überstand. </li><li> Wash Harz 5x mit 1 ml Puffer A: Invert Top-über-Boden, bis das Harz suspendiert, sammeln Harz durch Zentrifugation bei niedriger Geschwindigkeit, und entfernen Sie den Überstand. </li><li> Aussetzen Harz in 1 ml Puffer A und aliquotiert sie in der Anzahl der Rohre entsprechenden Proben zu verarbeiten. Sammeln Harz durch Zentrifugation bei niedriger Geschwindigkeit, und entfernen Sie den Überstand. Hinweis: Mischen Sie das Harz auch durch Umkehren Top-über-Boden. </li><li> Add 950 ul der verbleibenden WCE (aus Schritt 2.11: Extraktion der Proteine) zu 25 &amp; mgr; l des Harzes. </li><li> Inkubieren auf einer Schüttelplattform bei 4 ° C über Nacht. </li><li> Zentrifuge bei 800-1500 x g für 1 Minute bei 4 ° C. </li><li> Sparen Sie 50 ul als Überstand (SUP). Dann werden 50 ul 2x Laemmli Probenpuffer und Inkubation bei 100 ° C in einem Heizblock für 3-5 min. Load 10 ul jeder Probe in einem SDS-PAGE-Gel für Western Blot-Analyse (Abschnitt 5: Western-Blot-Analyse). </li><li> Wash Harz 5x mit 1 ml Puffer A: Invert Top-über-Boden, bis das Harz suspendiert, sammeln Harz durch Zentrifugation bei niedriger Geschwindigkeit, und entfernen Sie den Überstand. </li><li> Resuspendieren des Harzes in 100 ul SUMEB Probenpuffer (Tabelle 1). Hinweis: SUMEB Probenpuffer enthält 8 M Harnstoff und 1% SDS (starke Denaturierung Bedingung). </li><li> Inkubation bei 100 ° C in einem Heizblock für 3-5 min. </li><li> Vortex, dann bei 13.000 xg zentrifugieren für 30 Sekunden. Den Überstand in ein frisches Röhrchen. </li><li> Last 10-20 ul jeder Probe in einem SDS-PAGE-Gel für Western Blot-Analyse (Abschnitt 5: Western-Blot-Analyse). Anmerkung: 10-20 ul entspricht 0,5-1,0 mg Eingang, wenn 5 mg WCE verwendet. </li></ol> 5. Western Blot-Analyse <ol><li> Laden Sie die Proteinextrakte auf 4-12% Bis-Tris-Gelen (Table of Materials) und perform Elektrophorese bei 120 V für 90 Minuten (Laufpuffer: Table of Materials). </li><li> Übertragen Sie die Proteine ​​aus Gel auf eine Nitrocellulosemembran (Table of Materials) mit einem Übertragungsgerät bei 30 V für 90 min (Transfer-Puffer: Table of Materials). </li><li> Blockieren die Membran in 5% Milch / 1x TBST (Tabelle 1) für 1 h bei Raumtemperatur. </li><li> Inkubieren Membran mit dem primären Antikörper (Table of Materials) in 5% Milch / 1x TBST über Nacht bei 4 ° C. Für primäre Antikörper, verwenden Sie eine Verdünnung von 1: 1000 (Anti-Flag und Anti-Ub-Antikörper) oder 1: 5000 (Anti-Myc-Antikörper). </li><li> Für jeweils 10 min mit 1x TBST waschen Membran 3x. </li><li> Inkubieren Membran mit Sekundärantikörper (Table of Materials) in 5% Milch / 1x TBST für 1 Stunde bei Raumtemperatur. Verwenden Sie eine 1: 5000 Verdünnung des sekundären Antikörper. </li><li> Für jeweils 10 min mit 1x TBST waschen Membran 3x. </li><li> Inkubieren Membran mit ECL working-Lösung (Table of Materials) für 5 min. </li><li> Setzen Sie die Membran auf blauempfindlichen Röntgenfilm (Table of Materials) und die Entwicklung unter Verwendung einer automatischen Entwickler (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.

Epitop-Tags, wie HA, Myc, Flagge, und GST sind weit verbreitet für die biochemische Analyse von Proteinen verwendet. Konstruktion von Stämmen mit HF-Smt3 und Myc-markierten kinetochore Proteine ​​wie Ndc10 und Ndc80, erleichtert den Nachweis von PTMs wie sumoylation und Ubiquitinierung. HF-Smt3 Pulldown Assay erlaubt den Nachweis von sumoyliert Kinetochor-Proteinen, Ndc10 und Ndc80 (Abbildung 1). Der Affinitätsreinigungsprotokoll und Western Blot-Analyse unter Verwendung von Anti-Flag-Antikörper...

Ubiquitinierung und sumoylation ermöglicht die Konjugation von Ubiquitin and Small Ubiquitin-like Modifier (SUMO; Smt3 in S. cerevisiae 1) an ein Zielprotein auf. PTMs der Kinetochor-Proteinen beeinflussen ihre zellulären Ebenen und Protein-Protein-Interaktionen in verschiedenen Zellzyklusphasen treu Chromosomensegregation zu gewährleisten. Beispielsweise sind Zellebenen Cse4 / CENP-A und äußeren Kinetochorenprotein DSN1 durch Ubiquitin-vermittelte Proteolyse zur Sicherstellung Genomstabilität 2-5 geregelt. Destabilisierung der falsche Kinetochor-Mikrotubuli-Anhänge erfordert die Ipl1 / Aurora B-Kinase, die Dam1 und Ndc80 Komplexe, die direkt mit den Mikrotubuli interagieren 6-8 phosphoryliert. Obwohl die Identifizierung von mehr als siebzig kinetochore Proteinen gibt es sehr wenige Untersuchungen, die die Modifikationen dieser Proteine ​​mit PTMs zB Ubiquitin und SUMO untersuchen. Eine wichtige Einschränkung ist die Fähigkeit, die PTM zu bewahrens während der Reinigung und dem Mangel an individuelle Antikörper zum Nachweis von PTMs wie sumoylation, Phosphorylierung, Methylierung, und andere. Charakterisierung von sumoyliert Kinetochor-Proteinen Ndc10, Cep3, genutzt Bir1 und Ndc80 eine benutzerdefinierte Antikörpers 9. Zusätzlich hat Ndc10 als Substrat für Ubiquitinierung 10 in Verbindung gebracht. Menschliche Hec1 (Ndc80 in S. cerevisiae) wird auch für die Ubiquitinierung Substrat durch APC / C-hCdh1 E3-Ligase 11 geregelt. Daher Ndc10 und Ndc80 sind gute Kandidaten für die Optimierung des Protokolls sowohl sumoylation und Ubiquitinierung in S. erkennen cerevisiae. Um die Identifizierung von Sumoylierung zu erleichtern, konstruierten wir Stämme, die HF-Smt3 und Myc-tagged Ndc10 oder Ndc80 auszudrücken. Die Verwendung von Epitop-Tags (HF: His6-Flag) minimiert den Hintergrund aufgrund von Kreuzreaktivität, die häufig in polyklonales Serum gegen einen Kandidaten-Protein erzeugt wurden beobachtet. Wir entwickelten ein Protokoll zur Affinitätsreinigen HF-Smt3 Konjugate und dann verwendet kommerziellen anti-Flag und Anti-Myc-Antikörper, die Anwesenheit von sumolyated Ndc10 und Ndc80 im gereinigten Präparat Smt3 detektieren. Für Ubiquitinierung, entwickelten wir eine modifizierte Immunpräzipitationsprotokoll die Ubiquitinierung des Myc-tagged Kinetochor-Proteinen konserviert und Western Blot-Analyse durchgeführt, mit kommerziellen anti-Ub-Antikörper gegen Ubiquitinierung von Ndc10 und Ndc80 zu erkennen.

<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

Post-translationale Modifikationen (PTMs), wie Phosphorylierung, Methylierung, Acetylierung, Ubiquitinierung und sumoylation, regulieren die zelluläre Funktion von vielen Proteinen. PTMs der Kinetochor-Proteinen, die mit Centromer-DNA assoziieren vermitteln treu Chromosomensegregation auf Genomstabilität zu gewährleisten. Biochemischen Ansätzen wie Massenspektrometrie und Western-Blot-Analyse werden am häufigsten für die Identifizierung von PTMs verwendet. Dabei wird ein Protein-Reinigungsverfahren beschrieben, das den Nachweis von sowohl sumoylation und Ubiquitinierung des kinetochore Proteinen Ndc10 und Ndc80, in Saccharomyces cerevisiae ermöglicht. Ein Stamm, der zum Ausdruck bringt Polyhistidin-Flag-markierten Smt3 (HF-Smt3) und Myc-tagged Ndc10 oder Ndc80 wurde gebaut und für unsere Studien verwendet. Für den Nachweis von Sumoylierung erarbeiteten wir ein Protokoll zur Affinitätsreinigung His-Tag sumoyliert Proteinen durch Verwendung von Nickel Perlen und verwendet Western Blot-Analyse mit Anti-Myc-Antikörper zu erkennen sumoyliert Ndc10 einnd Ndc80. Für den Nachweis von Ubiquitinierung, entwickelten wir ein Protokoll für die Immunpräzipitation von Myc-markierte Proteine ​​verwendet und Western-Blot-Analyse mit anti-Ub-Antikörper zeigen, dass Ndc10 und Ndc80 werden ubiquitinierten. Unsere Ergebnisse zeigen, dass Epitop-markiertes Protein von Interesse in der His-Flag markiert Smt3 Stamm erleichtert den Nachweis mehrerer PTMs. Künftige Studien ermöglichen sollten Ausnutzung dieser Technik zur Identifizierung und Charakterisierung Protein-Wechselwirkungen, die abhängig von einem bestimmten PTM sind.

Zu sumoylation von kinetochore Proteine ​​Ndc80 und Ndc10 detektieren Stämme mit HF-Smt3 und Myc-markierten kinetochore Proteine ​​(Ndc80 oder Ndc10) konstruiert (Tabelle 2), wie zuvor beschrieben, 9,12. HF-Smt3 Konjugate wurden Affinität mit Ni-NTA-Perlen gereinigt. Western Blot-Analyse von gereinigtem HF-Smt3 mit einem Anti-Flag-Antikörper erlaubt Detektion sumoylierten Formen SUMO Substrate, die in dem Kontrollstamm ohne HF-Smt3 (1A und 1B, link...

Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

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

Diese Handschrift beschreibt den Nachweis von Sumoylierung und Ubiquitinierung von Kinetochor-Proteinen, Ndc10 und Ndc80, in der Bäckerhefe Saccharomyces cerevisiae.

Protein Purification Technik, die Erkennung von Sumoylierung und Ubiquitinierung von Bäckerhefe Kinetochor Proteine ​​Ndc10 und Ndc80 Erlaubt

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 Aufrufe.  National Cancer Institute, National Institute of Health.  Diese Handschrift beschreibt den Nachweis von Sumoylierung und Ubiquitinierung von Kinetochor-Proteinen, Ndc10 und Ndc80, in der Bäckerhefe Saccharomyces cerevisiae. 

Serie: 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.

Mess-replikative Lebensspanne in der Hefe

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

Forschung

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.

Detektion von Protein 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.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

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.

Reinigung von Mitochondrien aus Hefezellen

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.

Die Analyse der Entwicklung eines morphologischen Phänotyp als Funktion der Protein-Konzentration in Hefezellen

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.

Reinigung von Hsp104, ein 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 ...

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.

Chromatographische Reinigung von hoch aktiven Hefe Ribosomen

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.

Acquiring Fluoreszenz Zeitraffer-Filme von Bäckerhefe und Analysieren von Einzel-Zell-Dynamics mit Transplantaten

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.

Bäckerhefe Protein Extraktion und Reinigung für das Studium der Funktion, Wechselwirkungen, und post-translationale Modifikationen

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-basierte Screening Expression von Membranproteinen 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 ...

Protein Purification Technik, die Erkennung von Sumoylierung und Ubiquitinierung von Bäckerhefe Kinetochor Proteine ​​Ndc10 und Ndc80 Erlaubt

Protein Purification Technik, die Erkennung von Sumoylierung und Ubiquitinierung von Bäckerhefe Kinetochor Proteine Ndc10 und Ndc80 Erlaubt