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.

Maya Hücreleri 1. Büyüme <ol><li> Küçük bir kap içinde YPD (Tablo 1) 30 ml maya hücreleri inoküle. Çalkalama ile 30 ° C&#39;de gece boyunca inkübe edin. </li><li> YPD 50 ml, 600 nm (OD 600 = 0.2) ile 0.2 &#39;lik bir optik yoğunluğa kadar hücreleri seyreltilir ve çalkalanarak 30 ° C&#39;de inkübe edin. </li><li> 600 nm (OD 600 = 1.0), 1.0&#39;lik bir optik yoğunluğa kadar kültür büyütün. </li><li> Santrifüj hücreleri 2,000 xg&#39;de 5 dakika boyunca ve supernatant atın. </li><li> Hücreleri yıkamak için 2000 x g&#39;de 5 dakika boyunca 40 ml steril su ve santrifüj hücre pelletini. Süpernatant atılır ve -20 ° C &#39;de hücre pelletini saklayın. </li></ol> Proteinlerin 2. Ekstraksiyon <ol><li> Ya da Ni-NTA Superflow boncuklar kullanılarak açılır deneyi için buzla soğutulmuş guanidin tamponu (Tablo 1) 0.5 ml olarak yeniden süspanse hücreleri, immüno-çökeltme için (Tablo 1) tampon maddesi. Her zaman buz üzerinde tüpler tutun. </li><li> 2 m transferl vidalı kapaklı tüp. </li><li> Cam boncuklar aynı hacimde (Malzeme Tablo) ekleyin. </li><li> Daha sonra 2-3 dakika süreyle buz koyun, oda sıcaklığında 2 dakika süreyle mini boncuk çırpıcı hücreleri (Malzeme Tablo) Boncuk yendi. Bu üç kez tekrarlayın. </li><li> 30-60 dakika boyunca 4 ° C &#39;de yüksek hızda vorteksleyin. Hücreler lize sağlamak için mikroskop altında görselleştirme hücreleri kontrol edin. Not: Liz olmuş hücreler gibi karanlık hayaletler görünür ve bir sınır veya tanımlanmış şekli yoksun olacak. En uygun olarak, hücrelerin en azından% 80 lize edilmelidir. </li><li> 15 ml konik tüp (vidalı kapak gevşek olmalıdır) bir koleksiyonda bir itme pimi ve yer kullanarak tüpün dibinde bir delik delmek. </li><li> Lizat toplamak için 1 dakika için 1000 x g&#39;de santrifüjleyin. </li><li> Bir mikro santrifüj tüpüne lizat aktarın. </li><li> 4 ° C&#39;de 30 dakika boyunca 15,000 x g&#39;de santrifüjleyin ekstre proteinleri toplamak. </li><li> Protein assa kullanılarak ekstre proteinlerin konsantrasyonu ölçüny kiti (Malzeme Tablo) ve tüm özler normale protein aynı miktarda içermesi. Uygun bir tampon ile 1 ml toplam hacmi getirmek. Not: toplam proteinin yaklaşık 5 ila 50 mg OD 600 hücrelerinden elde edilir. </li><li> Bütün hücre ekstresi (WCE) halinde (adım 2.10 çıkarılan toplam protein, 5 mg olduğunda, 250 ug protein), 50 ul kaydedin. 2x Laemmli numune tampon maddesi (Tablo 1) 50 ul ilave edin ve 3-5 dakika için bir ısı blokta 100 ° C de inkübe edilir. Yük Western Blot analizi (Bölüm 5: Western blot analizi) bir SDS-PAGE jel içinde her numune için 10 ul. </li></ol> HF-Smt3 Konjugatlarının 3. saflaştırılması <ol><li> Deney 1 dakika boyunca düşük hızda santrifüj leme (800-1,500 xg) (örnek başına boncuk 100 ul) için gerekli olan Ni-NTA Superflow boncuk (Malzemelerin Tablo) elde edilir. Süpernatantı. </li><li> Yıkama boncuk PBS (Malzeme Tablosu) 1 ml 5 kat: Invert üst aşırıboncuklar yeniden süspanse kadar alt düşük hızda santrifüj boncuk toplamak ve süpernatant kaldırmak. </li><li> Guanidin tamponu (Tablo 1) 1 ml boncuk askıya alma ve işlenecek olan örneklere karşılık gelen boruların sayısı içine kısım. Düşük hızda santrifüj ile boncuk toplayın ve süpernatant kaldırmak. Üst-over tersini alt tarafından iyi boncuk karıştırın. Not: Üst-over altını ters çevirerek iyice boncuk karıştırın. </li><li> Ni-NTA Superflow boncuk 100 ul: her bir numune için, (protein çıkarması Aşama 2.11) geri kalan BOA&#39;da 950 ul ilave edin. </li><li> En az 4 saat veya gece boyunca 4 ° C &#39;de, bir sallanan platform üzerinde inkübe edin. </li><li> 4 ° C&#39;de 1 dakika için 800-1,500 xg&#39;de santrifüj. </li><li> Süpernatant (SUP) olarak 50 ul kaydedin. 2x Laemmli numune tampon maddesi 50 ul ilave edin ve 3-5 dakika için bir ısı blokta 100 ° C de inkübe edilir. Yük Western Blot Analizi (Kısım 5 için bir SDS-PAGE jel içinde her numune için 10 ul: Batı benek AnalYsis). </li><li> Sallanan bir platform üzerinde bir kez 5-10 dakika süreyle guanidin tampon maddesi içinde 1 ml yıkama boncuk. </li><li> Yıkama boncuklar sallanan bir platform üzerinde 5-10 dakika süreyle tampon maddesi (Tablo 1) kırılması için 1 ml 5 kat. </li><li> 2x Laemmli numune tampon maddesi 90 ul içinde süspanse boncuklar. </li><li> 1 M imidazol, 10 ul ekle. Not: İmidazol bağlı imidazol ve halkalar arasında rekabet etkileşim Ni-NTA boncuklardan His-etiketli proteini ayırmak için yardımcı olur. </li><li> 3-5 dakika için bir ısı blokta 100 ° C&#39;de inkübe edilir. </li><li> Girdap, daha sonra 30 saniye boyunca 13,000 x g&#39;de santrifüjlenir. Yeni bir tüp süpernatant aktarın. </li><li> (: Western blot analizi Bölüm 5) Western blot analizi için bir SDS-PAGE jel içinde her bir numunenin yük 10-20 ul. Not: BOA&#39;da 5 mg kullanıldığı takdirde, 10-20 ul, giriş 0.5-1.0 mg karşılık gelir. </li></ol> Myc-etiketli Kinetokor Proteinlerin 4. immünopresipitasyonu <ol><li> Anti-c-myc agaroz afinite jel, bir elde edilirtibody (Malzeme Tablo) düşük hızda santrifüj (800-1,500 xg) deneylerinde (örnek başına reçine 25 ul) için gerekli. Süpernatantı. </li><li> Tampon A ile 1 ml &#39;si ile yıkayınız reçinesi 5x: Reçine yeniden süspansiyon haline kadar ters çevirin en fazla alt düşük hızlı santrifüjleme ile reçine toplamak ve supernatant çıkarın. </li><li> Tampon A içinde 1 ml bir reçine askıya alma ve işlenecek olan örneklere karşılık gelen boruların sayısı içine kısım. Düşük hızlı santrifüjleme ile reçine toplamak ve supernatant çıkarın. Not: Üst-over-bottom tersini de reçineyi karıştırın. </li><li> Reçine 25 ul (protein ekstraksiyonu Aşama 2.11) geri kalan BOA&#39;da 950 ul ekle. </li><li> Gece boyunca 4 ° C &#39;de, bir sallanan platform üzerinde inkübe edin. </li><li> 4 ° C&#39;de 1 dakika için 800-1,500 xg&#39;de santrifüj. </li><li> Süpernatant (SUP) olarak 50 ul kaydedin. 2x Laemmli numune tampon maddesi 50 ul ilave edin ve 3-5 dakika için bir ısı blokta 100 ° C de inkübe edilir. BakReklam Western blot analizi için bir SDS-PAGE jel (Bölüm 5: Western blot analizi) &#39;de her bir numune için 10 ul. </li><li> Tampon A ile 1 ml &#39;si ile yıkayınız reçinesi 5x: Reçine yeniden süspansiyon haline kadar ters çevirin en fazla alt düşük hızlı santrifüjleme ile reçine toplamak ve supernatant çıkarın. </li><li> SUMEB numune tamponu (Tablo 1), 100 ul reçine yeniden süspanse edin. Not: SUMEB örnek tamponu 8 M üre ve% 1 SDS (güçlü denatüre koşul) içerir. </li><li> 3-5 dakika için bir ısı blokta 100 ° C&#39;de inkübe edilir. </li><li> Girdap, daha sonra 30 saniye boyunca 13,000 x g&#39;de santrifüjlenir. Yeni bir tüp süpernatant aktarın. </li><li> (: Western blot analizi Bölüm 5) Western blot analizi için bir SDS-PAGE jel içinde her bir numunenin yük 10-20 ul. Not: BOA&#39;da 5 mg kullanıldığı takdirde, 10-20 ul, giriş 0.5-1.0 mg karşılık gelir. </li></ol> 5. Western Blot Analizi <ol><li> % 4-12 Bis-Tris jelleri (Malzeme Tablo) ve perf üzerinde protein özleri yükleyinorm Elektroforez 90 dakika boyunca 120 V (tampon Running: Malzemelerin Tablosu). </li><li> 90 dakika boyunca (: Malzemelerin Tablo transfer tampon maddesi) 30 V bir transfer aparatı kullanılarak bir nitroselüloz zar (Malzemelerin Tablo) jelden proteinleri aktarın. </li><li> Oda sıcaklığında 1 saat boyunca% 5 süt / TBST 1x (Tablo 1) membran bloke eder. </li><li> % 5 süt / TBST 1x gece boyunca 4 ° C birincil antikor (Malzeme Tablo) ile membran inkübe edin. 1000 (anti-Flag ve anti-Ub antikorlar) ya da 1: 5000 (anti-myc antikoru), birincil antikor, bir 1 seyreltme kullanın. </li><li> 1x TBST ile 10 dakika her biri için membran 3x yıkayın. </li><li> Oda sıcaklığında 1 saat boyunca% 5 süt / TBST içinde 1 x sekonder antikor (Malzemelerin Tablo) ile membran inkübe edin. Sekonder antikor 5,000 seyreltme: 1 kullanın. </li><li> 1x TBST ile 10 dakika her biri için membran 3x yıkayın. </li><li> ECL worki ile membran inkübeng çözeltisi (Malzeme Tablo) 5 dakika. </li><li> Mavi duyarlı X-Ray film membranı (Malzeme Tablo) Açığa ve otomatik geliştirici (Malzeme Tablosu) kullanarak geliştirmek. </li></ol>

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The authors have nothing to disclose.

HA, Myc, Bayrak ve GST edildiği gibi epitop işareti yaygın proteinlerin biyokimyasal analizi için kullanılmıştır. HF-Smt3 ve Ndc10 ve Ndc80 Myc-etiketli kinetochore proteinler ile suşların inşaatı, örneğin sumoylation ubikuinasyon olarak PTMS tespitini kolaylaştırır. HF-Smt3 tahlil aşağı çekme sumoylated kinetochore proteinler, Ndc10 ve Ndc80 (Şekil 1) algılanmasını sağlar. Anti-Flag antikor kullanılarak afınite saflaştırma protokolü ve western lekeleme analizi, HF-Smt3 ve...

Sırasıyla bir hedef proteine ​​(S. cerevisiae 1 Smt3 SUMO) yayılmasını ve sumoylation ubikitin ve küçük Ubikuitin benzeri değiştirici konjügasyonunu sağlar. Kinetochore proteinlerinin PTMS sadık kromozom segregasyonu sağlamak için farklı bir hücre döngüsü aşamaları sırasında hücresel düzeyleri ve protein-protein etkileşimlerini etkiler. Örneğin, Cse4 / CENP-A ve dış kinetochore proteini Dsn1 hücresel düzeyleri genom stabilitesini 2-5 sağlanması için ubikuitin-aracılı proteolizi ile düzenlenir. Yanlış kinetochore-mikrotübül ekleri destabilizasyonu doğrudan mikrotübül 6-8 ile etkileşim Dam1 ve Ndc80 kompleksleri fosforile Ipl1 / Aurora B kinaz gerektirir. Yetmiş üzerinde kinetochore proteinlerin belirlenmesi rağmen, PTMS, örneğin ubikitin ve SUMO ile bu proteinlerin değişiklikler araştırmak çok az sayıda çalışma vardır. Büyük bir sınırlama PTM korumak için yeteneğisaflaştırma ve sumoylation, fosforilasyon, metilasyon, ve diğerleri gibi PTMS tespiti için özel antikorların yetersizliği s üzerine çıkar. Sumoylated kinetochore proteinlerin karakterizasyonu Ndc10, Cep3, Bir1 ve Ndc80 özel bir antikor 9 kullanılmaktadır. Buna ek olarak, Ndc10 ubikitinasyon 10, bir alt-tabaka olarak dahil edilmiştir. İnsan Hec1 (S. cerevisiae&#39;deki Ndc80), APC / Cı-hCdh1 E3 ligaz 11 düzenlenir, ubikitinasyon için maddedir. Bu nedenle, Ndc10 ve Ndc80 S. sumoylation ve ubikitinasyona hem algılamak için protokol optimizasyonu için iyi adaylardır cerevisiae. Sumoylation belirlenmesini kolaylaştırmak için, HF-Smt3 ve Myc etiketli Ndc10 veya Ndc80 ifade eden suşları inşa. epitop etiketleri (HF: His6-Bayrak) kullanılması nedeniyle sık, bir aday proteine ​​karşı poliklonal serum içinde gözlenir çapraz reaktivite arka en aza indirir. Biz afinite bir protokol geliştirdiHF-Smt3 birleşikleri saflaştırılması ve daha sonra saf hale Smt3 hazırlanmasında sumolyated Ndc10 ve Ndc80 varlığını tespit etmek için, ticari bir anti-Flag ve anti-myc antikoru kullanılır. Ubikitinasyon sağlamak için, Myc-etiketli kinetochore proteinlerinin dağılmasını koruyan bir tadil edilmiş immüno protokolü tasarlanmış ve Ndc10 ve Ndc80 ubikitinasyonuna tespit etmek için ticari bir anti-Ub antikoru ile Western Blot analizi gerçekleştirilmiştir.

<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

Fosforilasyon, metilasyon, asetilasyon, ubikitinasyon ve sumoylation gibi post-translasyonel modifikasyonlar (PTMS), pek çok protein, hücresel fonksiyonu düzenleyen. Sentromerik DNA ile ilişkilendirmek kinetochore proteinlerin PTMS genom istikrarı korumak için sadık kromozom tecrit aracılık eder. Bu tür kütle spektrometrisi ve western blot analizi gibi biyokimyasal yaklaşımlar en yaygın PTMS tanımlanması için kullanılır. Burada, bir protein saflaştırma yöntemi Saccharomyces cerevisiae&#39;de kinetochore proteinler, Ndc10 ve Ndc80, bir sumoylation ubikuinasyon hem saptanmasına izin veren tarif edilmektedir. Ndc10 Myc-etiketli ya Ndc80 inşa ve çalışmaları için kullanılmıştır ifade polihistidin-Flag-işaretli Smt3 (HF-Smt3) ve A suşu. Sumoylation saptanması için, nikel boncuklar kullanılarak His ile etiketlenmiş sumoylated proteinleri saflaştırmak için bir protokol geliştirilmiştir ve sumoylated Ndc10 a tespit etmek için bir anti-myc antikor ile Western blot analizi kullanılannd Ndc80. Ubikitinasyon saptanması için, Myc-etiketli proteinlerin immüno için bir protokol geliştirilmiştir ve Ndc10 ve Ndc80 ubikitinlenmiş olduğunu göstermek için anti-Ub antikor ile Western blot analizi kullanılmıştır. Bizim sonuçlarımız His-Bayrak ilgi epitop etiketli protein Smt3 suşu birden PTMS tespitini kolaylaştırır etiketlenmiş olduğunu göstermektedir. Gelecekteki çalışmalar bu tekniğin sömürü belirli bir PTM bağımlı protein etkileşimleri belirlemek ve tanımlamak için izin vermelidir.

Daha önce tarif edildiği gibi 9,12 kinetochore proteinleri Ndc80 ve Ndc10 bölgesinin sumoylation tespit etmek için, HF-Smt3 ve Myc-etiketli kinetochore proteinleri (Ndc80 veya Ndc10) suşlar, (Tablo 2) yapılmıştır. HF-Smt3 konjugatları afinite Ni-NTA boncuklar kullanılarak saflaştırılmıştır. HF-Smt3 (Şekil 1A ve 1B, sol panel) içermeyen kontrol suşu yoktu SUMO substratların sumoylated formları, bir anti-Bayrak antikor izin algılama ile a...

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

Bu yazıda çiçek mayası Saccharomyces cerevisiae içinde, sumoylation ve kinetochore proteinleri, Ndc10 ve Ndc80 ubikitinasyonuna bağlanmasını anlatır.

Sumoylation ve geliştirici Maya Kinetokor Proteinler Ndc10 ve Ndc80 ve ubikuitinasyomınun Tespiti verir Protein Saflaştırma Tekniği

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 Görüntüleme.  National Cancer Institute, National Institute of Health.  Bu yazıda çiçek mayası Saccharomyces cerevisiae içinde, sumoylation ve kinetochore proteinleri, Ndc10 ve Ndc80 ubikitinasyonuna bağlanmasını anlatır. 

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

Measuring Replicative Life Span in the Budding Yeast

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

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

Geliştirici Maya replikatif Ömrü Ölçüm

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

Biyoloji

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.

Protein Ubikitinasyon Tespiti

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.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

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.

Maya hücreleri Mitokondri saflaştırılması

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.

Geliştirici Maya Protein Konsantrasyonu bir işlev olarak Morfolojik Fenotip Kalkınma Analizi

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.

Hsp104 saflaştırılması, 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.

Yüksek Aktif Maya Ribozomlar Kromatografik Arıtma

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.

Tomurcuklanma Maya Floresan Time-lapse Filmler Kazanılması ve GREFTLERİ kullanarak Tek hücreli Dynamics analiz

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.

Fonksiyon Çalışma, Etkileşimler, ve Post-translasyonel değişiklikler için tomurcuklanan Maya Protein Ekstraksiyonu ve Saflaştırılması

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.

Membran proteinlerinin Yeşil floresan proteini temelli Ekspresyon elemesinde<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 ...