Biz kütle spektrometresi analizleri ve yararlı tartışmalar ile teknik yardım için teşekkür Dr Zengin Rogers, ve Dr. Rob Moritz, Jeff Ranish ve yararlı tartışmalar için Hamid Mirzai. Bu çalışma, NIH Mükemmeliyet Merkezleri tarafından finanse edildi.

Hücre büyümesi ve medya <ol><li> 1.0 minimal bir maya orta iki (Amonyum Sülfat veya amino asitler olmaksızın% 0.17 Maya Azot Bankası,% 0.5 &#39;lik Amonyum sülfat) 1 litre kültürü içine sonra tohum OD 600 100 mL zengin medya geceleme BY4742Δarg4Δlys1 hücreleri tek bir koloni içeren bir (Isotec 13 C 6 15 N 4) ve lisin (13 C 6 15 N 2; Isotec) 20 mg / L isotopically normal veya ağır arginin ile desteklenmiş amino asitler, tam. </li><li> Hücreler 1,8 OD 600, 18 saat boyunca yetiştirilmiştir. Etiketli izotopların tam birleşme hücreleri elde etmek için en az 9 nesiller geçmesi kritik öneme sahiptir. Işık örnek, orta (isotopically Normal arginin ve lizin,% 0.2 oleat (Sigma Kimyasallar) ve% 0.5 Tween 40 (Sigma Kimyasallar)) içeren bir oleat içine reseeded ve başka bir 85 dakika için uyarılan, steril su ile yıkanır, pelet. Bu arginin ve lizin açısından bir isotopically ışık verimleri, örnek ve isotopically ağır bir glikoz yetişen referans örnek oleat uyarılır. </li></ol> Hücre Lizis, İzolasyon ve Peptitler, Fraksiyonu <ol start="3"><li> Santrifüj şişesi tartılır. 3 dakika boyunca santrifüj Hasat örnekleri. Medya çıkarın ve aşırı sıvı aspire. Pelet ve şişe (genellikle 1-2 gram arasında verim) sıvı nitrojen sonra flaş dondurma tartılır. &#39;Taşlama&#39; tampon pelet ağırlığı, sıvı azot (Fosfat% 10 gliserol, Proteaz İnhibitörleri (SigmaFAST Proteaz İnhibitör Tablet, Sigma, (PBS, Gibco) Tuzlu Tamponlanmış) dondurulmuş pelet hacmi eşdeğer ekleyin ve Fosfataz inhibitörleri (Thermo Scientific HALT )). </li><li> Taşlama damar sıvı nitrojen içinde dondurun. Sıvı azot kaynama geçene kadar bekleyin. Dondurulmuş bilyeli rulmanlar, taşlama gemi pelet aktarın. </li><li> 600 rpm&#39;de 3 dakika için bir yön ters ile 1 dk 20 sn döngüsü ile Grind. Sıvı nitrojen içinde taşlama gemi dondurursam. Toplam öğütme süresi 15 dakika 4 kez daha tekrarlayın. </li><li> 50ml Falcon tüp yeri kuru buz içine donmuş grindate toplayın. Kullanmak üzere saklayın -80 ° C ye kadar hazır. </li><li> 8M üre, 0.1M amonyum bikarbonat, 0.1M Tris pH: 8.6 10ml çözüm olun. Üre tampon 3 cilt donmuş grindate 1 hacim (örneğin üre tampon 3mls için 1 ml PBS ile 1 gram pelet) ekleyin. Hemen bir prob ucu sonikatör (10 saniye bakliyat 2) ile karışım sonikasyon. Ucu çözüm köpük önlemek için tüpün alt kısmına yakın tutun. Grindate hemen çözüm gitmek gerekir. </li><li> 4 5 dakika santrifüj lizat temizleyin ° C </li><li> Supernatant taze tüplerine transfer. </li><li> 0,5 M Tris yeni bir kısım olun [2-carboxyethyl] fosfin (TCEP). 5mM bir final konsantrasyon için 1:100 örnek ekleyin. 37 ° C&#39;de 1 saat </li><li> Azaltılmış cysteines Alkilasyon. Oda sıcaklığına kadar soğumasını bekleyin. 1M iodoacetamide taze bir kısım olun. (Tüp folyo ile sarın) karanlıkta tutun. 20mm bir final konsantrasyon ekle. Karanlıkta, oda sıcaklığında 1 saat süreyle inkübe edin. </li><li> 1 saat boyunca oda sıcaklığında 1M stok 20mm DTT iodoacetamide Quench </li><li> Protein standart Bradford veya BCA assay (tarif) ile sayısal olarak. Aşağıdaki SDS PAGE ile analiz için bir örnek ele alalım. </li><li> Ağır izotoplar dahil doğrulamak için, azaltılmış ve alkillenmiş lizat 100 ul kullanın. DH 2 O ile 01:04 sulandırınız sonikasyon ve tripsin için 50:1 protein tripsin eklemek. C18 Ultramicrospin sütunlar (The Nest Grubu) ile elektrikli süpürge ve sonra desalt hız aşağı en az 4 saat, kuru örnek Digest. <ol><li> 100 ul Asetonitril Hidrat sütun </li><li> 200 ul% 0.1 TFA ile dengelenmesi. </li><li> 200 ul% 0.1 TFA kuru örnek tekrar Sütun üzerine yükleyin. ~ 200 xg veya sütun akışı için gerekli minimum hız santrifüjleyin. </li><li> 200 ul% 0.1 TFA ile iki kez yıkayın. </li><li> 50 ul Asetonitril% 60,% 0.1 TFA ile iki kez ile Zehir. </li><li> Vac hız Kuru örnek. </li><li> 20 ul% 0.1 formik asit süspanse edin örnek. Bir kütle spektrometresi 2 ul çalıştırın. Isotopically ağır arginin ve ilgili m / z spektrumları 10 ve 8 Dalton vardiya tarafından lizin dahil kontrol edin. % 98 - 96 arasında her amino asit için birleşme olmalıdır. </li></ol></li><li> Isotopically ağır ve hafif örneklerin eşdeğer miktarda (protein mg) karıştırın. </li><li> % 20 metanol ile örnek 01:04 sulandırınız. </li><li> 1:200 az tripsin ekleyin. 37 o gece inkübe ° C </li><li> SDS SAYFA sindirim tamamlanması için gitti. 2 ve 4 predigestion örnek ul ve 8 çalıştırınd 16 ul sonrası tripsin sindirmek. Coomassie boyama sindirim eksiksiz olduğunu gözünüzde canlandırın. Tam değilse, bir prob ucu sonikatör kısaca örnek sonikasyon ve tripsin ile sindiremez. </li><li> Vac hız örnek kurulayın. Kuru pelet çözüm girer kadar süzülmüş dH 2 O ve vorteks 200 ul eklediğinizde. Vac bir hızda aşağı Kuru örnek. DH 2 O ile yeniden süspanse edin Örnek yeniden kurulayın. </li><li> % 0.1 Trifluoracetic Asit yeniden süspanse pelet (TFA, Sigma). PH pH şeritleri kullanarak 3 civarında olduğunu kontrol edin. % 10 TFA ile asitlenir Gerekirse. </li><li> Pelet dışında herhangi bir çökelti. </li><li> Waters Sep-Pak Vac 500 mg C18 kolon örnekleri Desalt. Bu sütunlar aşırı yüklenmeyin. Maksimum miktarı 5 mg ama biraz daha az yük bulunuyor. Örnek gerekirse birden çok sütun arasında Böl. <ol><li> 5 ml% 100 asetonitril Hidrat sütun. ~ 200 xg veya sütun akışı için gerekli minimum hız santrifüjleyin. </li><li> 5ml% 0.1 TFA ile sütun dengelenmesi. Örnek Yük. Akışını toplamak ve yeniden yükleyin. </li><li> 5 ml% 0.1 TFA ile yıkayınız. Tekrarlayın. </li><li> Zehir 2ml 60% Asetonitril,% 0.1 TFA. </li><li> Vac bir hızda aşağı Kuru örnek. </li></ol></li></ol> Phosphopeptides Peptid Fraksiyonu ve İzolasyon <ol><li> Peptitler HILIC bir sütun üzerinde fraksiyone. Biz Tosoh TSK-Gel Amid-80 4.6 mm x 25 cm bir bekçi sütun ile analitik kolon. Kullanın Bu adım için zaman ayırın, sonra çalıştırmak iki boş gradyanlar numunenin yüklemeden önce% 90 az 2 saat sütun çalıştırmak. XX etrafında bu boyutu olduğu düşünülen bir sütun üzerinde maksimum yük, genellikle başına en fazla 5 mg vadede yük yoktur. </li><li> Solvent A -% 98 ACN/0.1% TFA Solvent B -% 2 ACN/0.1% TFA % 90 yük üzerinde 20 &#39;,% 90,% 85 üzerinden 5,% 85% 60 A üzerinden 40,% 60% 0 5 üzerinden&#39;% 0,% 90 A üzerinde 5 &#39;. </li><li> Akış hızı 1ml/min ve fraksiyonları% 85% 60 A degrade başlayarak 2 dakika aralıklarla toplanmıştır. </li><li> 10 kesirler sayısını azaltmak için kesirler birleştirin. Artan kesirler muhtemelen phosphoproteome daha fazla kapsama alanı verecektir. </li></ol> Phosphopeptides, zenginleştirilmesi <ol><li> Phosphopeptides PHOS seçin Demir Affinity Jel ile zenginleştirilmiştir. Biz kısım tekrarlanır dondurulmasından kaçınmak için elimizden IMAC reçine. 50 ml 250 mM asetik asit,% 30 ACN yük çözümü olun. HCl ile pH 2.7 &#39;ye getirin. 500 ul yük çözüm pelet tekrar HCl veya NaOH ile ayarlamak gerekli olduğu, pH kontrol edin. </li><li> Ön yıkama 200 uygun bir sütun IMAC boncuk ul. Biz Molbiotec dönüş sütunları kullanın. Her fraksiyonu% 50 bulamaç (jel çözüm yüklemek için) 15 ul ekleyin ve sonunda dönme üzerinde son 30 örnekleri &#39;kuluçkaya yatmaktadır. Analizi için akış tutun. </li><li> Yük çözüm örnekleri 3 kere yıkayın ve 500 kez ile ul süzülmüş dH 2 O </li><li> 2 Zehir peptidler - 400 50 mM Na 2 HPO 4 (pH 8.4) tampon ul ile 3 dk. Bir cam flakon aktarın. </li><li> Ile pH 3 ila 5 ul% 100 Formik asit, sıvı azot flaş dondurma ve hız vac kurutulmuş numune aşağı asitlenir. </li><li> % 0.1 TFA ve yukarıda açıklandığı gibi temiz C18 200 ul süspanse edin phosphopeptides. Vac hız yıkandı, örnekleri kurulayın. </li><li> % 0.1 formik asit 20 ul peptitler tekrar 2 kütle spektrometresi analizleri başına ul ya da gerektiği gibi kullanın. </li></ol> Notlar Bu protokol için &#39;kör&#39; bir çalışma olacak. Hücre parçalama batı blot ve Bradford ve BCA deneyleri ve tripsin sindirim da kolaylıkla tespit edilebilir, izlenebilir. HILIC sütun bu benzer bir iz verdi, ya da diğerleri [1, 2] bağlı olarak kullanılan likit kromatografi makine tarafından görülebilir. Örnekler kütle spektrometresi tarafından izlenmekte; ucuz makineleri gibi yüksek bir kitle doğruluğu, yüksek çözünürlüklü makine yüksek maliyet analizleri gitmeden önce LCQs veya MALDIs gibi örnekleri test edin. Bir asit ile bir örnek kurutma Son olarak, cam kullanın.

<ol>
	<li>Albuquerque, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multidimensional+chromatography+technology+for+in-depth+phosphoproteome+analysis.">A multidimensional chromatography technology for in-depth phosphoproteome analysis.</a> Mol Cell Proteomics. 7 (7), 1389-1396 (2008).</li><li>McNulty, D. E., Annan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+chromatography+reduces+the+complexity+of+the+phosphoproteome+and+improves+global+phosphopeptide+isolation+and+detection.">Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.</a> Mol Cell Proteomics. 7 (5), 971-980 (2008).</li><li>Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags.">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nat Biotechnol. 17 (10), 994-999 (1999).</li><li>Ross, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Gruhler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteomics+applied+to+the+yeast+pheromone+signaling+pathway.">Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.</a> Mol Cell Proteomics. 4 (3), 310-327 (2005).</li><li>Wilson-Grady, J. T., Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+fission+yeast.">Phosphoproteome analysis of fission yeast.</a> J Proteome Res. 7 (3), 1088-1097 (2008).</li><li>Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCX/IMAC+enrichment+approach+for+global+phosphorylation+analysis+by+mass+spectrometry.">The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.</a> Nat Protoc. 3 (10), 1630-1638 (2008).</li><li>Jensen, S. S., Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+impact+of+some+experimental+procedures+on+different+phosphopeptide+enrichment+techniques.">Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.</a> Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).</li><li>Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns.">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Mol Cell Proteomics. 4 (7), 873-886 (2005).</li><li>Pinkse, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+at+the+femtomole+level+of+phosphopeptides+from+proteolytic+digests+using+2D-NanoLC-ESI-MS/MS+and+titanium+oxide+precolumns.">Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.</a> Anal Chem. 76 (14), 3935-3943 (2004).</li><li>Tao, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteome+analysis+using+a+dendrimer+conjugation+chemistry+and+tandem+mass+spectrometry.">Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.</a> Nat Methods. 2 (8), 591-598 (2005).</li><li>Zhou, H., Watts, J. D., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+to+the+analysis+of+protein+phosphorylation.">A systematic approach to the analysis of protein phosphorylation.</a> Nat Biotechnol. 19 (4), 375-378 (2001).</li><li>Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+isolation+of+distinct,+overlapping+segments+of+the+phosphoproteome.">Reproducible isolation of distinct, overlapping segments of the phosphoproteome.</a> Nat Methods. 4 (3), 231-237 (2007).</li></ol>

Bu yöntem, kantitatif analiz edilebilir phosphopeptides bir zenginlik verecektir. Parametrelerin sayısı bu protokolde değişiklik, ama hatırlanması gereken en önemli yönü, fosforilasyon korumak için olabilir. Eğer böyle ICAT gibi in vivo, etiketleri hücrelerin etiket değil. Eğer örneğin ölçümü için hücrelerin hazırlanması çok sayıda farklı şekillerde elde olması. [3 veya ITRAC [4] farklı ölçümü için iki kültürler etiketlemek için yazılan çıkarma kullanılır olması. et...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Isotec&trade; L-Arginine-13C6,15N4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>608033</td>
<td>&nbsp;</td>
<tr>
<td>Isotec&trade; L-Lysine-15N2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>609021</td>
<td>&nbsp;</td>
<tr>
<td>GIBCO&#x2122; Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>70011044</td>
<td>&nbsp;</td>
<tr>
<td>SigmaFAST Protease Inhibitor Tablets</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Pierce Halt Phosphatase Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>78420</td>
<td>&nbsp;</td>
<tr>
<td>Retsch PM100 Ball Mill Grinder </td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>20.540.0001</td>
<td>&nbsp;</td>
<tr>
<td>Retsch 125 ml Stainless Steel Grinding Jar</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>01.462.0148</td>
<td>&nbsp;</td>
<tr>
<td>Ultramicrospin C18 column</td>
<td>&nbsp;</td>
<td>The Nest Group</td>
<td>SUM SS10</td>
<td>&nbsp;</td>
<tr>
<td>Sep-Pak Vac 500 mg C18</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT043395</td>
<td>&nbsp;</td>
<tr>
<td>TSK-Gel Amide-80 4.6 mm x 25 cm analytical column</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>13071</td>
<td>This is the HILIC chromatographic column.</td>
</tr>
<tr>
<td>Guard column 4.6 mm ID x1 cm</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>19021</td>
<td>We recommend this guard column to extend the life of your HILIC column.</td>
</tr>
<tr>
<td>PHOS-Select&trade; Iron Affinity Gel</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P9740</td>
<td>This is the IMAC resin. Aliquot and store.</td>
</tr>		</tbody>
		</table>
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quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

Bu protokol isotopically ağır ve hafif S. cerevisiae hücrelerinin yağ asidi oleat, büyüme ve stimülasyon açıklar. Hücreler bir bilyalı değirmen değirmeni ve üre çözünürleştirme çözüm getirilen grindate cryolysis prosedür kullanılarak öğütülür. Bu prosedür, fosforilasyon korunması ve hücre parçalama sırasında phosphoproteome reorientation önlenmesi, metabolik olarak inaktif bir devlet hücreleri lizis sağlar. Azaltma, alkilleme tripsin, proteinlerin sindirimi takiben, örnekleri C18 sütun ve fraksiyonasyon hidrofilik etkileşim kromatografi (HILIC) kullanılarak azaltılabilir örnek karmaşıklığı desalted. HILIC sütun tercihen phosphoproteomics için de uygundur hidrofilik molekülleri korur. Fosforile peptidler kromatografik profili olmayan fosforile muadillerine göre daha sonra Zehir eğilimindedir. Fraksiyonasyon sonra, phosphopeptides phosphopeptide zenginleştirme için ücret tabanlı yakınlık dayanmaktadır immobilize metal kromatografi kullanılarak zenginleştirilmiştir. Bu prosedürün sonunda örneklerin kantitatif kütle spektrometresi ile analiz edilmesi için hazırız.

Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Cryolysis, üre solubilziation, HILIC fraksiyonasyon ve fosforile peptidlerin IMAC zenginleştirme kullanarak kantitatif fosforilasyon prosedürü açıklaması.

Yağ Asidi Kantitatif Phosphoproteomics Uyarılmış Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

quantitative-phosphoproteomics-fatty-acid-stimulated-saccharomyces

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Biology

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

10.0K Görüntüleme.  Institute for Systems Biology. Cryolysis, üre solubilziation, HILIC fraksiyonasyon ve fosforile peptidlerin IMAC zenginleştirme kullanarak kantitatif fosforilasyon prosedürü açıklaması.

Video: Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Diseksiyonu Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Biyoloji

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Mikroarray Analizi için Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Maya İzleme Mitophagy Floresan Mikroskopi Testi Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

HO-Endonuclease Oluşum kantitasyonu ve Analiz Tavlama Tek-Strand kromozomal Translokasyonlar Uyarılmış Saccharomyces cerevisiae</em

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

İzlenmesi protein refolding için birleştiğinde Tahliller<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

MRNA Çürüme Oranlarının Ölçümü<em&gt; Saccharomyces cerevisiae</emKullanma&gt;<em&gt; Rpb1-1</em&gt; Suşlar

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Kimyasal Genetik Etkileşimleri Hızlı Kimlik<em&gt; Saccharomyces cerevisiae</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Evrim Yöntemi yönettiği<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Kütüphane Oluşturma ve Tarama

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).