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. Crescimento de Células de Levedura <ol><li> Inoculação das células de levedura em 30 ml de YPD (Tabela 1) em um balão pequeno. Incubar a 30 ° C durante a noite com agitação. </li><li> Dilui-se as células até uma densidade óptica de 0,2 a 600 nm (DO 600 = 0,2) em 50 ml de meio YPD e incubar a 30 ° C com agitação. </li><li> Crescer a cultura a uma densidade óptica de 1,0 a 600 nm (DO600 = 1,0). </li><li> Centrifugar células durante 5 min a 2000 xg e desprezar o sobrenadante. </li><li> Ressuspender o sedimento celular em 40 ml de água estéril e centrifugar durante 5 minutos a 2000 x g para lavar as células. Descartar o sobrenadante e o sedimento de células armazenar a -20 ° C. </li></ol> 2. Extracção de proteínas <ol><li> Ressuspender as células em 0,5 ml de tampão de guanidina arrefecido em gelo (Tabela 1) para o ensaio suspenso usando pérolas de Ni-NTA Superflow ou tampão A (Tabela 1) para imunoprecipitação. Manter tubos em gelo em todos os momentos. </li><li> Transferir a 2 ml tubo de tampa de rosca. </li><li> Adicionar o mesmo volume de esferas de vidro (Tabela de Materiais). </li><li> Conta-bater as células de uma mini-grânulo batedor (tabela de materiais) durante 2 min à temperatura ambiente, depois colocar sobre gelo durante 2-3 minutos. Repita isso três vezes. </li><li> Vortex em alta velocidade, a 4 ° C durante 30-60 min. Verificar as células por visualização sob o microscópio para garantir que as células são lisadas. Nota: As células lisadas aparece como fantasmas escuros e não têm um limite ou uma forma definida. Optimamente, pelo menos, 80% das células devem ser lisadas. </li><li> Perfurar um buraco no fundo do tubo usando um pino do impulso e coloque em uma coleção 15 ml tubo cônico (tampa de rosca deve ser solto). </li><li> Centrifugar a 1.000 xg durante 1 min para recolher o lisado. </li><li> Transferir o lisado para um tubo de micro centrífuga. </li><li> Centrifugar a 15.000 xg durante 30 min a 4 ° C para recolher as proteínas extraídas. </li><li> Medir a concentração das proteínas extraídas utilizando a proteína assay kit (Tabela de Materiais) e normalizar todos os extractos de conter a mesma quantidade de proteína. Levar o volume total a 1 ml com tampão apropriado. Nota: Cerca de 5 mg de proteína total é obtida a partir de 50 OD600 células. </li><li> Guardar a 50 ul (250 ug de proteína, se a proteína total extraído a partir do passo 5 é 2,10 mg) como extracto celular total (WCE). Adicionar 50 ul de tampão de amostra Laemmli 2x (Tabela 1) e incuba-se a 100 ° C num bloco de aquecimento durante 3-5 minutos. Carga de 10 ul de cada amostra num gel de SDS-PAGE para análise de Western Blot (Secção 5: Análise de Western blot). </li></ol> 3. Purificação de HF-Smt3 Conjugados <ol><li> Obter os grânulos Superflow Ni-NTA (Tabela de Materiais) necessários para os experimentos (100 ul de contas por amostra) por centrifugação a baixa velocidade (800-1,500 xg) durante 1 min. Remover o sobrenadante. </li><li> Grânulos lavagem 5x com 1 ml de PBS (Tabela de Materiais): Inverter top-sobre-inferior até que as contas são novamente suspensas, coletar pérolas por centrifugação a baixa velocidade, e remover o sobrenadante. </li><li> Suspender pérolas em 1 ml de tampão de guanidina (Tabela 1) e aliquotar-los para o número de tubos correspondentes a amostras a serem processadas. Coletar pérolas por centrifugação a baixa velocidade, e remover o sobrenadante. Misture as contas bem invertendo top-over inferior. Nota: Misture bem grânulos invertendo top-over inferior. </li><li> Para cada amostra, adicionar 950 ul de WCE remanescente (a partir do Passo 2.11: Extracção de proteínas) com 100 ul de contas de Ni-NTA Superflow. </li><li> Incubar numa plataforma de agitação a 4 ° C durante pelo menos 4 horas ou durante a noite. </li><li> Centrifugar a 800-1,500 xg durante 1 min a 4 ° C. </li><li> Economize 50 ul como sobrenadante (SUP). Adicionar 50 ul de tampão de amostra Laemmli 2x e incuba-se a 100 ° C num bloco de aquecimento durante 3-5 minutos. Carga de 10 ul de cada amostra num gel de SDS-PAGE para análise de Western Blot (Secção 5: Anal Western blotysis). </li><li> Lavar as pérolas uma vez com 1 ml de tampão de guanidina durante 5-10 min sobre uma plataforma oscilante. </li><li> Lavar as pérolas de 5x com 1 ml de tampão de Rebentamento (Tabela 1) durante 5-10 minutos numa plataforma de agitação. </li><li> Grânulos Ressuspender em 90 ul de tampão de amostra Laemmli 2x. </li><li> Adicionar 10 ul de 1 M de imidazol. Nota: Imidazole ajuda para dissociar a proteína marcada com His a partir das pérolas de Ni-NTA, devido à interacção entre a competir imidazole e os grânulos. </li><li> Incubar a 100 ° C num bloco de aquecimento durante 3-5 minutos. </li><li> Vortex, em seguida, centrifugar a 13.000 xg durante 30 seg. Transferir o sobrenadante para um tubo fresco. </li><li> Carga 10-20 ul de cada amostra num gel de SDS-PAGE para análise de Western Blot (Secção 5: Análise de Western blot). Nota: 10-20 ul corresponde a 0,5-1,0 mg de entrada, se 5 mg de EOC é usado. </li></ol> 4. Immunoprecipitation de Myc-marcados kinetochore Proteínas <ol><li> Obter anti-c-Myc de afinidade em gel de agarose umtibody (Tabela de Materiais) necessários para os experimentos (25 ul de resina por exemplo) por centrifugação a baixa velocidade (800-1,500 xg). Remover o sobrenadante. </li><li> Lavar 5x resina com 1 mL de tampão A: Inverter cima-baixo-cima até que a resina é ressuspensa, recolher resina por centrifugação a baixa velocidade, e remover o sobrenadante. </li><li> Suspender a resina em 1 ml de tampão A e aliquotar-los para o número de tubos correspondentes a amostras a serem processadas. Recolha resina por centrifugação a baixa velocidade, e remover o sobrenadante. Nota: Misture a resina bem invertendo-bottom top-over. </li><li> Adicionar 950 ul da WCE remanescente (a partir do Passo 2.11: Extracção de proteínas) com 25 ul de resina. </li><li> Incubar numa plataforma de agitação a 4 ° C durante a noite. </li><li> Centrifugar a 800-1,500 xg durante 1 min a 4 ° C. </li><li> Economize 50 ul como sobrenadante (SUP). Adicionar 50 ul de tampão de amostra Laemmli 2x e incuba-se a 100 ° C num bloco de aquecimento durante 3-5 minutos. Load 10 uL de cada amostra num gel de SDS-PAGE para análise de Western Blot (Secção 5: Análise de Western blot). </li><li> Lavar 5x resina com 1 mL de tampão A: Inverter cima-baixo-cima até que a resina é ressuspensa, recolher resina por centrifugação a baixa velocidade, e remover o sobrenadante. </li><li> Ressuspender a resina em 100 ul de tampão de amostra SUMEB (Tabela 1). Nota: tampão de amostra contém SUMEB Ureia 8 M e 1% de SDS (forte condição de desnaturação). </li><li> Incubar a 100 ° C num bloco de aquecimento durante 3-5 minutos. </li><li> Vortex, em seguida, centrifugar a 13.000 xg durante 30 seg. Transferir o sobrenadante para um tubo fresco. </li><li> Carga 10-20 ul de cada amostra num gel de SDS-PAGE para análise de Western Blot (Secção 5: Análise de Western blot). Nota: 10-20 ul corresponde a 0,5-1,0 mg de entrada, se 5 mg de EOC é usado. </li></ol> 5. Análise de mancha Ocidental <ol><li> Coloque os extratos de proteínas em 4-12% Bis-Tris géis (Tabela de Materiais) e perfeletroforese orm a 120 V durante 90 minutos (tampão corrente: Tabela de Materiais). </li><li> Transferir as proteínas do gel para uma membrana de nitrocelulose (Tabela de Materiais) usando um aparelho de transferência a 30 V durante 90 min (tampão de transferência: Tabela de Materiais). </li><li> Bloquear a membrana em 5% leite / TBST 1x (Tabela 1) durante 1 h à temperatura ambiente. </li><li> Incubar membrana com anticorpo primário (Tabela de Materiais) em 5% leite / TBST 1x durante a noite a 4 ° C. Para os anticorpos primários, usar uma diluição de 1: 1000 (os anticorpos anti-FLAG e anti-Ub) ou 1: 5000 (anticorpo anti-Myc). </li><li> Lavar 3x membrana durante 10 min cada com TBST 1x. </li><li> Incubar membrana com anticorpo secundário (Tabela de Materiais) em 5% leite / TBST 1x durante 1 hora à temperatura ambiente. Usar uma diluição de 1: 5000 de anticorpos secundários. </li><li> Lavar 3x membrana durante 10 min cada com TBST 1x. </li><li> Incubar membrana com ECL workisolução ng (Tabela de Materiais) durante 5 min. </li><li> Expor a membrana sensível ao azul filme X-Ray (Tabela de Materiais) e desenvolver usando um desenvolvedor automático (Tabela de Materiais). </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. 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The authors have nothing to disclose.

Marcadores de epitopo HA, tais como, Myc, Bandeira, e GST são amplamente utilizados para análise bioquímica de proteínas. Construção de estirpes com IC-Smt3 e proteínas cinetócoro Myc-tag, como Ndc10 e Ndc80, facilita a detecção de PTMs como sumoilação e ubiquitinação. HF-Smt3 puxar para baixo ensaio permite a detecção de proteínas cinetócoro sumoylated, Ndc10 e Ndc80 (Figura 1). O protocolo de purificação por afinidade e análise Western blot utilizando anticorpo anti-Flag estabelec...

Ubiquitinação e sumoilação permitir a conjugação de ubiquitina e modificador pequeno Ubiquitina-like (SUMO; Smt3 em S. cerevisiae 1) para uma proteína-alvo, respectivamente. PTMs de proteínas cinetócoro afectar os níveis celulares e interacções proteína-proteína durante as diferentes fases do ciclo celular para assegurar a segregação cromossómica fiel. Por exemplo, os níveis celulares de Cse4 / CENP-A e proteína cinetócoro exterior Dsn1 são regulados através de proteólise mediada por ubiquitina para assegurar a estabilidade do genoma 2-5. A desestabilização das ligações cinetócoro-microtúbulo incorrectas requer a IPL1 / Aurora B-quinase, que fosforila e Dam1 Ndc80 complexos que interagem directamente com os microtúbulos 6-8. Apesar da identificação de mais de setenta proteínas cinetócoro, há muito poucos estudos que investigam as modificações dessas proteínas com PTMs, por exemplo, ubiquitina e SUMO. Uma limitação importante é a capacidade de preservar a PTMs durante a purificação e a escassez de anticorpos para a detecção de mercadorias, tais como PTMs sumoilação, fosforilação, metilação, e outros. Caracterização de proteínas cinetócoro sumoylated Ndc10, Cep3, Bir1, e Ndc80 utilizou um anticorpo costume 9. Além disso, Ndc10 tem sido implicado como um substrato para ubiquitinação 10. Hec1 Humano (Ndc80 em S. cerevisiae) é também substrato para ubiquitinação, regulamentada pelo APC / C-hCdh1 E3 ligase 11. Portanto, Ndc10 e Ndc80 são bons candidatos para otimização do protocolo para detectar tanto sumoylation e ubiquitination em S. cerevisiae. Para facilitar a identificação de sumoilação, construiu-se as estirpes que expressam HF-Smt3 e marcada com myc Ndc10 ou Ndc80. O uso de marcadores de epitopo (HF: His6-Flag) minimiza o fundo devido à reatividade cruzada que é frequentemente observada no soro policlonal criado contra uma proteína candidato. Eu inventei um protocolo de afinidadepurificar conjugados HF-Smt3 e, em seguida, utilizado anti-Flag comercial e anticorpos anti-myc para detectar a presença de sumolyated Ndc10 e Ndc80 na preparação Smt3 purificado. Para ubiquitination, eu inventei um protocolo de imunoprecipitação modificado que preserva ubiquitinação das proteínas cinetócoro Myc marcadas e realizadas análises de Western blot com o anticorpo anti-Ub comercial para detectar ubiquitination de Ndc10 e Ndc80.

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

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

As modificações pós-traducionais (PTMs), tais como fosforilação, metilação, acetilação, ubiquitinação, e sumoilação, regular a função de muitas proteínas celulares. PTMs de proteínas cinetócoro que se associam com DNA centromeric mediar segregação cromossômica fiéis para manter a estabilidade do genoma. Abordagens bioquímicas, tais como espectrometria de massa e análise de transferência de western são mais comumente utilizado para a identificação de PTMs. Aqui, um método de purificação de proteínas está descrito que permite a detecção de ambos sumoilação e ubiquitinação das proteínas cinetocoro, Ndc10 Ndc80, e em Saccharomyces cerevisiae. Uma estirpe que expressa polihistidina-Flag-etiquetado Smt3 (HF-Smt3) e marcada com myc Ndc10 ou Ndc80 foi construída e utilizada para os nossos estudos. Para a detecção de sumoylation, eu inventei um protocolo para purificar por afinidade proteínas sumoylated His-tag usando esferas de níquel e usado western blot com anti-Myc anticorpo para detectar sumoylated Ndc10 umnd Ndc80. Para a detecção de ubiquitinação, que concebido um protocolo para a imunoprecipitação das proteínas Myc-tag e utilizada a análise de Western blot com um anticorpo anti-Ub para mostrar que Ndc10 e Ndc80 são ubiquitinadas. Os nossos resultados mostram que o epitopo marcado com proteína de interesse na sua bandeira-tagged Smt3 estirpe facilita a detecção de vários PTMs. Estudos futuros deverão permitir a exploração desta técnica para identificar e caracterizar as interações proteína que são dependentes de um PTM específico.

Para detectar sumoilação de proteínas cinetócoro Ndc80 e Ndc10, estirpes com HF-Smt3 e proteínas cinetócoro marcada com myc (Ndc80 ou Ndc10) foram calculados (Tabela 2), como anteriormente descrito 9,12. HF-Smt3 conjugados foram purificados por afinidade utilizando esferas de Ni-NTA. A análise de Western blot de HF purificado-Smt3 com um anticorpo anti-Flag permitiu a detecção de formas sumoylated de substratos SUMO que estavam ausentes na estirpe de controlo, sem HF-Smt3 (Fi...

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

Este manuscrito descreve a detecção de sumoylation e ubiquitinação de proteínas cinetócoro, Ndc10 e Ndc80, na levedura Saccharomyces cerevisiae brotamento.

Técnica de purificação de proteína que permite a detecção de SUMOilação e Ubiquitination de brotamento Yeast kinetochore Proteínas Ndc10 e Ndc80

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

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

Research

JoVE Journal

Biology

12.0K visualizações.  National Cancer Institute, National Institute of Health.  Este manuscrito descreve a detecção de sumoylation e ubiquitinação de proteínas cinetócoro, Ndc10 e Ndc80, na levedura Saccharomyces cerevisiae brotamento. 

Vídeo: 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.

Medição Span vida replicativa na levedura de brotamento

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

Biologia

Detection of Protein Ubiquitination

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

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

Detecção de Ubiquitinação Protein

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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.

Purificação de mitocôndrias de células de levedura

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.

Análise do desenvolvimento de um fenótipo morfológico como uma função da concentração de proteínas em levedura de brotamento

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.

Purificação da Hsp104, um Disaggregase Protein

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.

Purificação cromatográfica de Ribossomos Yeast Highly Active

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

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

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

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

Aquisição de fluorescência Filmes Time-lapse da levedura de brotamento e análise da dinâmica de células simples utilização de enxertos

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.

Brotando Yeast extração e purificação de proteínas para o estudo da função, interações e modificações pós-traducionais

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.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<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 ...