Name: protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80
Uploaded: 2015-05-03T14:00:00
Description: 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

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. Vækst af gærceller <ol><li> Inokulering gærceller i 30 ml YPD (tabel 1) i en lille kolbe. Der inkuberes ved 30 ° C natten over under omrystning. </li><li> Fortynd cellerne til en optisk densitet på 0,2 ved 600 nm (OD600 = 0,2) i 50 ml YPD og inkuberes ved 30 ° C under omrystning. </li><li> Vokse kulturen til en optisk densitet på 1,0 ved 600 nm (OD600 = 1,0). </li><li> Centrifuger cellerne i 5 minutter ved 2.000 xg og kassere supernatanten. </li><li> Resuspender cellepe...

<ol>
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Epitopmærker såsom HA, Myc, Flag, og GST er almindeligt brugt til biokemisk analyse af proteiner. Konstruktion af stammer med HF-Smt3 og Myc-mærkede kinetochore proteiner, såsom Ndc10 og Ndc80, letter påvisning af PTMS såsom sumoylation og ubiquitination. HF-Smt3 trække ned assay muliggør detektion af sumoylated kinetochore proteiner, Ndc10 og Ndc80 (figur 1). Affinitetsoprensning protokol og Western blot-analyse under anvendelse af anti-Flag-antistof fastslå specificiteten af ​​interaktion...

Ubiquitinering og sumoylation tillader konjugation af ubiquitin og Lille Ubiquitin-lignende modifier (SUMO; Smt3 i S. cerevisiae 1) til et målprotein, hhv. PTMS af kinetochore proteiner påvirker deres cellulære niveauer og protein-protein interaktioner under forskellige cellecyklus faser for at sikre trofaste kromosom adskillelse. For eksempel er cellulære niveauer af Cse4 / CENP-A og ydre kinetochore protein Dsn1 reguleret af ubiquitinmedieret proteolyse for at sikre genomstabilitet 2-5. Destabilisering af ukorrekte kinetochore-mikrotubulus vedhæftninger kræver IPL1 / Aurora B kinase, som phosphorylerer Dam1 og Ndc80 komplekser, direkte interagerer med mikrotubuli 6-8. På trods af den identifikation af over halvfjerds kinetochore proteiner, der er meget få undersøgelser, der undersøger de ændringer af disse proteiner med PTMS fx ubiquitin og SUMO. En væsentlig begrænsning er evnen til at bevare PTMs under oprensning og knapheden på brugerdefinerede antistoffer til påvisning af PTMS såsom sumoylation, phosphorylering, methylering, og andre. Karakterisering af sumoylated kinetochore proteiner Ndc10, Cep3, Bir1 og Ndc80 udnyttede en brugerdefineret antistof 9. Derudover har Ndc10 været inddraget som et substrat for ubiquitinering 10. Menneskelig Hec1 (Ndc80 i S. cerevisiae) er også substrat for ubiquitinering, reguleret af APC / C-hCdh1 E3 ligase 11. Derfor Ndc10 og Ndc80 er gode kandidater til optimering af protokollen til at afsløre både sumoylation og ubiquitinering i S. cerevisiae. For at lette identifikationen af ​​sumoylation, vi konstruerede stammer, der udtrykker HF-Smt3 og Myc-mærkede Ndc10 eller Ndc80. Anvendelsen af ​​epitopmærker (HF: His6-Flag) minimerer baggrunden på grund af krydsreaktivitet, der ofte observeres i polyklonalt serum rejst mod en kandidat-protein. Vi udtænkt en protokol til affinitetoprense HF-Smt3 konjugater og derefter anvendes kommercielle anti-Flag og anti-Myc-antistoffer til påvisning af tilstedeværelsen af ​​sumolyated Ndc10 og Ndc80 i oprenset Smt3 præparatet. For ubiquitinering, vi udtænkt en modificeret immunopræcipitering protokol, bevarer ubiquitinering af Myc-mærkede kinetochore proteiner og udførte western blot analyse med kommercielle anti-Ub antistof til at detektere ubiquitinering af Ndc10 og 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

Post-translationelle modifikationer (PTMS), såsom phosphorylering, methylering, acetylering, ubiquitinering, og sumoylation, regulere den cellulære funktion af mange proteiner. PTMS af kinetochore proteiner, der associerer med centromere DNA mediere trofast kromosomsegregering at opretholde genom stabilitet. Biokemiske metoder såsom massespektrometri og western blot analyse er mest almindeligt anvendt til identifikation af PTMS. Her er en proteinoprensning fremgangsmåde beskrevet som tillader påvisningen af både sumoylation og ubiquitinering af kinetochore proteiner, Ndc10 og Ndc80, i Saccharomyces cerevisiae. En stamme, der udtrykker polyhistidin-Flag-tagget Smt3 (HF-Smt3) og Myc-mærkede Ndc10 eller Ndc80 blev konstrueret og anvendes til vores studier. Til påvisning af sumoylation, vi udtænkt en protokol til affinitetsoprense His-mærket sumoylated proteiner ved hjælp af nikkel perler og brugte western blot analyse med anti-Myc-antistof til at detektere sumoylated Ndc10 ennd Ndc80. Til påvisning af ubiquitinering, vi udtænkt en protokol for immunopræcipitation af Myc-mærkede proteiner og brugte western blot analyse med anti-Ub antistof at vise, at Ndc10 og Ndc80 er ubiquitineret. Vore resultater viser, at epitop-tagget protein af interesse i His-Flag tagged Smt3 stamme letter påvisning af flere PTMS. Fremtidige undersøgelser bør give udnytte denne teknik til at identificere og karakterisere protein interaktioner, der er afhængige af en bestemt PTM.

For at detektere sumoylation af kinetochore proteiner Ndc80 og Ndc10, stammer med HF-Smt3 og Myc-mærkede kinetochore proteiner (Ndc80 eller Ndc10) blev konstrueret (tabel 2), som tidligere beskrevet 9,12. HF-Smt3 konjugater blev affinitetsoprenset under anvendelse af Ni-NTA-perler. Western blot analyse af oprenset HF-Smt3 med et anti-Flag-antistof tillod detektion af sumoylated former af SUMO substrater, der var fraværende i kontrolstammen uden HF-Smt3 (figur 1A og ...

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

Dette manuskript beskriver påvisning af sumoylation og ubiquitinering af kinetochore proteiner, Ndc10 og Ndc80, i spirende gæren Saccharomyces cerevisiae.

Proteinoprensning Teknik der tillader detektering af Sumoylation og Ubiquitinering af Spirende Gær Kinetochore Proteiner Ndc10 og Ndc80

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

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