Bu eser mali Kimyasal Tasarım (NRSC-Kataliz) tarafından Kontrollü Hollanda Ulusal Araştırma Okulu Kombinasyon, Catalysis bir araştırma bursu ile desteklenmiştir. Dr HY Steensma ve Leiden Üniversitesi&#39;nden Dr. GPH van Heusden S. rekombinant ifadesi ile desteklerinden dolayı kabul edilir S. cerevisiae Nar1.

Kur ve Çözümleri 1. Hazırlık <ol><li> 100 mM Tris, pH 8.5, 250 mM NaCI ve% 10 gliserol içeren tampon çözeltisi hazırlayın. Argon püskürtülerek tampon havasını. Oksijensiz bir bölme içine tampon tanıtılması. </li><li> Iki kısım halinde tampon bölün. Bir kısım (tampon A), 1 mM DL-ditiyotreitol eklemek ve 1 mM L-sistein (DTT, E 154,25 g / mol r). Diğer kısım (tampon B) indirgeyici maddeler içermez. </li><li> Oksidan ve indirgeyici çözümleri hazırlayın. 1 ml 2 çözeltileridir 20 ve 200 mM potasyum ferrisiyanür Yap (FIC, E 329,26 g / mol r) ve anaerobik tampon B içinde sodyum ditiyonit (SD, E 174,11 g / mol r) olarak </li><li> Aşağıdaki gibi arabulucu karışımı hazırlayın: <ol><li> B tampon aşağıdaki redoks mediatörleri ve 50 mM NaCI, her biri 160 uM ekleyin: N, N, N &#39;, N&#39; -tetrametil-p-phenylendediamine (TMPD, E 237,17 g / mol r, E &#39;0 0,276 V) , 2,6-diklorofenol İndofenol(DCIP, E 290,08 g / mol, E r, Metilen mavisi (E, r &#39;(0 0,055 V fenazin etosülfat 0 0,217 V) PES, E 334,4 g / mol, E r&#39;) 319,85 g / mol, bir E &#39;, 0 0,011 V) resorufin (M r 235,17 g / mol, bir E &#39;, 0 -0,051 V) Indigodisulphonate (M r 466,35 g / mol, bir E&#39;, 0 -0,125 V) 2-hidroksi-1,4-naphtaquinone ( M r 174,15 g / mol, bir E &#39;, 0 -0,145 V) antrakinon-2-sülfonat (M r 328,28 g / mol, E&#39; 0 -0,225 V) Phenosafranin (M r 322,8 g / mol, bir E &#39;, 0 -0,252 V), Safranin O (M r 350,84 g / mol, bir E &#39;, 0 -0,280 V) nötr kırmızı (M r 288,8 g / mol, E&#39; 0 -0,340 V) Benzil violojen (M r 409,4 g / mol, e &#39;0 -0,350 V) ve metil violojen (M 257,16 g / mol, E r&#39; 0 -0,440 V). </li><li> Arabulucu korumak için alüminyum folyo ile sarın şişeışıktan s. </li></ol></li><li> Hazırlama 10 mg / ml, S. cerevisiae strep etiketli Nar1 proteini tampon A Hızlı S. (181 uM, E 55.2 kDa r) E. S. cerevisiae Nar1 E. coli, bir strep etiketli protein olarak bir pET24A ifade vektörü kullanılmıştır. Strep-taktin kromatografi ortamı (IBA) ile bir anaerobik odasında proteini saflaştırmak ve Akta saflaştırıcısı (GE Healthcare) gibi bir protein saflaştırma sistemi kullanılarak saflaştırılması. </li><li> Aşağıdaki gibi bir anaerobik odasında redoks titrasyonu kurulumunu hazırlayın: <ol><li> Ag / AgCl referans elektrot ve titrasyon kabına bir platin tel elektrot bağlayın. V. kadar mV ölçebilen bir voltmetre hem elektrotlar bağlayın </li><li> Küçük bir karıştırma çubuğu yerleştirin. </li><li> Bir manyetik karıştırıcı üzerinde titrasyon kabı yerleştirin. </li><li> Tampon B, örneğin, 72 uM proteininin son konsantrasyonlara kadar 2 ml, ve her bir 38 uM aracı karışık çözeltisi ve protein çözeltisi ile titrasyon kabına doldurunArabulucu. Not: protein konsantrasyonu bağlı ve gerekli arabulucu ayarlanmalıdır üç çözümler miktarda konsantrasyonlar. </li><li> 30 dakika karıştırın ve voltmetre açın. </li></ol></li><li> 10 Temiz kuvars cam tüpler EPR hazırlayın. Havasız odadan tüpler tanıtılması. </li><li> Sıvı nitrojen ile bir küçük (200 ml yaklaşık) sıvı azot Dewar doldurun. </li></ol> 2. Redoks Titrasyonu gerçekleştirin <ol><li> Çözümün potansiyel stabil olana kadar bekleyin. Not: Pratikte bu potansiyel dakikada en az 1 mV sürüklenir anlamına gelir. </li><li> Voltmetre tarafından verilen potansiyeli not edin. </li><li> 200 ul ilk örnek çizin ve bir EPR tüpe enjekte. </li><li> Bir lastik tıpa ile tüp kap ve havasız odadan tüpü çıkarın. </li><li> Aşağıdaki gibi sıvı azot içinde tüp dondurma: Dikkat! Bu malzeme aksi durumda boru kıracak yavaşça yapılması gerekmektedir. <ol><li> İlk t eklemekSıvı azot içinde borunun ip. </li><li> Bir tıslama sesi duyulabilir kadar bekleyin. Not: Bu 10 saniye kadar sürebilir. </li><li> Örnek kısmı tamamen sular altında böylece yavaş yavaş tüp kalanını yerleştirin. </li></ol></li><li> -0.6 V çözeltinin potansiyelini düşürmek için titrasyon kabında çözeltiye, 2 mM SD bir çözelti küçük bir kısım 2/1 ul ekle Not: fazla 10 ul ilave edilmesi gerekir ise, 20 mM çözeltisine geçer. Belirli bir potansiyel değere terazide tartılır çok zor olduğundan, voltmetre kaydedilen gerçek değerler yazılı ve titrasyon eğrisini oluşturmak için kullanılır. </li><li> Aşama 2,3-2,5 tarif edildiği gibi bir numune alın. </li><li> Voltmetre tarafından verilen gerçek potansiyelini not edin. </li><li> Potansiyel circa 50 mV arttı kadar oksidan ekleyin. 2 mM FIC çözüm küçük bir kısım 1-2 ul ekleyerek başlayın. Birden fazla 10 ul ilave edilmesi gerekir ise, 20 mM çözeltisine geçer. </li><li> Ho aşağı Notw çözümleri çok eklenmiştir. Not: Bu titrasyon sırasında numune seyreltme için düzeltmek için gereklidir. </li><li> Adım 2,3-2,5 tarif edildiği gibi bir numune alın. </li><li> Voltmetre tarafından verilen gerçek potansiyelini not edin. </li><li> Tekrar kaplı olan -0.6 V 0,2 kadar V potansiyel aralığında kadar 2.9 ve 2.12 adımları ve 10 EPR tüpler 200 ul her doldurulur. Not: her çekilmesi ile hacmi azalır zamanda elektrot yerleştirme ayarlanması gerekir karıştırma müdahalesi olmadan çözelti ile iyi bir temas olabilir. Bu son örnek çekilmesi ve bu nedenle bu numunenin potansiyeli daha az doğru olabilir sonra potansiyel ölçmek zor olabilir. </li><li> EPR ölçümlerin gerçekleştirildiği kadar isteğe bağlı olarak, sıvı azot içinde dondurulmuş EPR örnekleri saklayın. </li></ol> EPR Spektroskopisi ve Veri Analizi Kullanılarak 3. Ölçü Titrasyon Örnekleri <ol><li> Farklı Rekor EPR spektrumlarıtitrasyon örnekleri şöyle: <ol><li> &lt;10 -5 çubuğuna kuvars kriyostat tahliye etmek için yüksek vakum pompası kullanma. </li><li> EPR mıknatısın su soğutma açın. EPR boşluğu yoluyla bir kuru hava akışını açın. </li><li> Mıknatıs güç kaynağı ve EPR bilgisayarda açın. </li><li> Bir frekans kalibrasyon programını çalıştırın. EPR ölçüm parametrelerini ayarlayın. Sıvı helyum termosa Kriyostat bağlayın. 9-16 K. boşluğu soğutun </li><li> Boşluğunda EPR örnek tanıtılması. EPR spektrumu kaydedin. </li></ol></li><li> Farklı EPR türlerin belirlenmesi ve sinyalleri ölçmek için en iyi spektrumları kullanın. Harici bakır standart çözüm spektrumu ile spektrumları ve karşılaştırma çift entegrasyonu ile EPR kantifikasyonunu Başarmak 10 mM CuSO / 2 M NaClO 4 4/10 mM HCl. 8 </li><li> Aşağıdaki gibi titrasyon eğrisini yapın: <ol><li> Para için karakteristik olan bir g-değeri ile EPR sinyali genliği çizilirpotansiyeline karşı ilgi manyetik türleri. </li><li> Bir spin için EPR sinyal genlik ölçeği değiştirme / molekül ölçeği adım 3.2 EPR kantifikasyonunu kullanarak. </li><li> Oksitleyici ya da indirgeyici ajan ilave nedeniyle meydana seyreltme için, her numunenin bir sinyal genliğini düzeltin. </li><li> Ag / AgCl referans elektrot orta noktası potansiyeli potansiyelini düzeltin. Not: (doymuş KCI) ile Ag / AgCİ referans elektrot, 25 ° C&#39;de standart bir hidrojen elektrotundan (SHE) karşı 0,194 V bir orta bir potansiyele sahiptir. Voltmetre potansiyel -0,6 V dolaylarında okur, bu nedenle, SHE referans potansiyel -0.4 V olduğunu </li></ol></li><li> Aşağıdaki gibi Nernst denklemi sinyali Fit: Bir türleri için bu azaltılmış durumda paramanyetiktir: <img alt="Denklem 1" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq1.jpg" width="200" /> Paramanyetik o olan bir tür içinxidized durumu: <img alt="Denklem 2" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq2.jpg" width="200" /> Bir ara madde halinde paramanyetik bir türleri için: <img alt="Denklem 3" fo:content-width="2.2in" src="/files/ftp_upload/51611/51611eq3.jpg" width="220" /> Bu denklemler bir elektron transfer edilir ki içinde redoks reaksiyonları için de geçerlidir, yani, n = 1. Volt E = potansiyeli E m = Volt orta potansiyeli F = Faraday sabiti = 96.480 ° C · mol -1 R = Gaz sabiti = 8,314 J · K -1 · mol -1 Kelvin T = Sıcaklık </li></ol>

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Yazarlar ifşa hiçbir şey yok.

13 redoks mediatör dizisi -0,45 gelen SHE karşı 0,3 V (Tablo 1 ve Şekil 3) aralığında çözelti içinde bir redoks denge sağlar. Peki yukarıda ve bu potansiyellerin altındaki tüm arabulucular ya tamamen azalır veya tamamen okside ve dolayısıyla protein redoks aktif merkezleri ile daha fazla dengeleme oluşabilir edilmektedir. Bazen redoks titrasyonları sadece dar bir aralık 11 ölçülecek izin, sadece iki ya da üç aracılarla gerçekleştirilen literatürde...

Bu gezegen, fotosentez, azot fiksasyonu ve solunum hayat sürdürme en temel kimyasal işlemler, organik ve inorganik redoks cofactors geniş bir yelpazede içeren büyük protein kompleksleri tarafından katalize edilir dikkat çekicidir. Tüm proteinlerin yaklaşık% 30 bir veya daha fazla metal kofaktörlerine içerdiğini tahmin edilmiştir. 1,2 belirlenmesi ve redoks kofaktör doğrudan elektrokimya (örneğin, protein filmi voltametri) veya redoks titrasyonlarını kullanılarak oluşturulabilir karakterize. İki teknikleri doğaları ve uygulanabilirliği tamamlayıcısıdır. Voltammetri bir elektrot yüzeyi ile reaksiyona girebilen orta potansiyelleri ve kofaktör elektron transferi kinetik hızlı belirlenmesi sunmaktadır. 3,4 Genellikle bu tür sitokrom-c veya ferrodoksin olarak elektron transfer proteinlerinin için iyi çalışır. Ve bazen bir elektrod yüzeyinde immobilize edilmiştir, daha karmaşık proteinler için de kullanılabilir. Detaylı bilgiprotein kofaktör doğası voltogramda kofaktör kimliğine ilişkin herhangi bir doğrudan bilgi vermez gibi, mevcut olmak zorundadır. Redoks titrasyonları yürütmek için daha zahmetli ve protein mg miktarlarda gerektirir. Ancak, orta potansiyeli ve kofaktör kimliği hakkında bilgi sunuyoruz. 5 Ayrıca, bir proteinin birden kofaktör izlenebilir bir tek Titrasyonda. Bir redoks titrasyonu prensibi redoks aktif protein veya enzim kimyasal azaltılmış ya da oksitlenmiş olmasıdır. Amacıyla kofaktör kullanılan indirgeyici veya oksidan redoks arabulucular ile reaksiyona emin olmak için. Bu redoks mediatörleri, çözeltiye potansiyel ölçülebilir şekilde bir elektrot ile reaksiyona girer. Arabulucular bir redoks tampon olarak hareket ve protein ve elektrot cofactors arasında dengeye. Kimyasal olarak, istenen değere potansiyel poising sonra, bir numune aw için sıvı azot içinde dondurulmuş hızlı bir şekilde çekilir vespektroskopik teknikleri ile daha fazla analiz AIT. EPR spektroskopisi bu miktar para-manyetik metal merkezi ya da organik radikalleri ölçmek için kullanılabilir bu açıdan özellikle yararlıdır. titrasyon iki yönde gerçekleştirilebilir: düşük yüksek ya da düşük yüksek orta olasılığı vardır. seçim çalışmanın altında kofaktörlerinin istikrar bağlıdır. Genellikle düşük potansiyelde başlayacak ve potansiyelini artırmak aşamalı oksidan eklemek akıllıca olacaktır. Bu oksidatif titrasyon denir ve burada tarif edilir. Burada Saccharomyces cerevisiae protein Nar1 içeren demir-sülfür küme redoks titrasyonu göstermektedir. Bu protein sitozolik demir-sülfür küme biyosentetik makineleri (CIA yolu) olarak, bir iskele protein olarak, büyük olasılıkla yer almaktadır. 6,7

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reference electrode (Ag/AgCl)</td>
			<td style="width:101px;">Radiometer</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemicals</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EPR spectrometer</td>
			<td style="width:101px;">Bruker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">N,N,N&#8217;,N&#8217;-tetramethyl-p-phenylendediamine (TMPD) &#183;(HCl)2</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">637-01-4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2,6-dichlorophenol indophenol (DCIP), sodium salt</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">620-45-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenazine ethosulfate (PES)</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">10510-77-7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene blue</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">122965-43-9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resorufin, sodium salt</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">34994-50-8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Indigodisulfonate (indigo carmine)</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">860-22-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-hydroxy-1,4-naphtaquinone</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">83-72-7</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Anthraquinone-2-sulfonate Na+ H2O</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">153277-35-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenosafranin</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">81-93-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Safranin O</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">477-73-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neutral red</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">553-24-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzyl viologen</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1102-19-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl viologen</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1910-42-5</td>
		</tr>
	</tbody>
</table>

epr monitored redox titration of the cofactors of saccharomyces cerevisiae nar1

Elektron Paramagnetik Rezonans (EPR) redoks titrasyonları güçlü bir yöntem proteinlerde kofaktörlerinin orta noktası potansiyelini belirlemek ve onların saptanabilir redoks devlet kofaktör tespit ve ölçmek için vardır izledi. teknik, elektron transfer oranları hakkında bilgi sunmuyor gibi, yaklaşımlar, ancak çalışılan protein kofaktör kimliği ve redoks devlet kurmak yok, doğrudan elektrokimya (voltametri) tamamlayıcı. tekniği bir elektron paramanyetik rezonans (EPR) tespit kofaktör içeren herhangi bir proteine ​​yaygın olarak uygulanmaktadır. Tipik bir titrasyon 1-100 uM aralığındaki bir kofaktör konsantrasyonu ile 2 ml protein gerektirir. proteinin belirli bir potansiyelde örnek poise için bir kimyasal indirgeyici (sodyum ditionit) veya oksidanın (potasyum ferrisiyanür) ile titre edilmiştir. Bir platin tel ve bir Ag / AgCl referans elektrot volt bağlımetre protein çözeltisi potansiyelini ölçmek için. 13 birçok farklı redoks mediatörü bir dizi protein redoks kofaktörler ve elektrotlar arasındaki denge için kullanılır. Numuneler protein farklı redoks kofaktörler için karakteristik farklı potansiyelleri ve Elektron paramanyetik rezonans spektrumları, çizilir ölçülür. Örnek potansiyeli karşı sinyal yoğunluğu arsa kofaktör orta noktası potansiyelini belirlemek amacıyla Nernst denklemi kullanılarak analiz edilir.

Nar1: Saccharomyces cerevisiae &#39;den bir protein Nar1 ihtiva eden bir demir-sülfür kümesinin titrasyon üç farklı kofaktörler tanımlanması sonucunu bir [3FE-4S] küme, bir [4Fe-4S] kümesi ve bir tek çekirdekli Fe merkezi (Şekil 1) . EPR sinyaller, G-değerleri ile karakterize edilir: için, [3FE-4S] + g Z 2.01 g çapraz 2.00 (Şekil 1B); için, [4Fe-4S] + g z 2.02 g y 1.93 x g 1,82 <stron...

Watch this Scientific Journal Video about EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1 at JoVE.com

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR kofaktörleridir Redox Titrasyonu İzlenen<em&gt; Saccharomyces cerevisiae</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

epr-monitored-redox-titration-cofactors-saccharomyces-cerevisiae

Research

JoVE Journal

Biology

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

13.4K Görüntüleme.  Delft University of Technology. The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

Video: EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

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 ...

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 ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Yılında Kistik Fibrozis Transmembran İletkenlik Regülatörü Protein İfadesi ve Arıtma Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

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 ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Olarak ifade edilmiştir kistik fibroz transmembran kondüktans regülatörü Protein Saflaştırılması<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

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.