We sincerely thank Thomas Howe and J&#246;rg Carsten for the programming and technical support with the liquid handling systems.

<ol>
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We have nothing to disclose.

Fenol-sülfürik asit yöntemle Polisakarit tespiti: Farklı monosakaridler bu yöntemin 9 kullanımı ile farklı bir absorpsiyon maksimum ve molar sönüm katsayılarını gösteriyor. Bu, farklı miktarlarda çeşitli şekerler içeren polimerlerden farklı soğurma maksimumları ile sonuçlanır. 16 farklı ticari olarak temin edilebilir polimerler için absorpsiyon maksimum seviyesinin farklı dalga boylarının Tablo 5&#39;de verilmiştir. Polimerler (1 g / L) içinde eritildi GKD 2 O (150 rpm), gece boyunca karıştırıldı ve fenol-sülfürik asit-yöntemi ile ölçülmüştür . Diutan sakız 470 nm ve skleroglukan ve hyaluronik asit 484 nm&#39;de en yüksek, en düşük maksimum absorpsiyon gösterdi. Bu sonuçlara dayanarak 480 nm bu tarama platformu için seçildi. Polimerlerin göreceli emme (% 100 olarak ayarlanmış), 1 g / L glikoz elde edilen absorbansa göre hesaplandı. düşük sonuçlar% 92, her ikisi de skleroglukan ve süksinoglikan ile elde edilmiştir. Bu ihr olduskleroglukan sadece glikoz içerir ve süksinoglikan 7 oranında glikoz ve galaktoz içerdiği yansıtılmaktadır: 1 arasındadır. Hem ticari polimerler kurutma ve farklı kül içeriğinin farklı kayıplar, bu ~% 110 teorik değeri ulaşılamamıştır nedeni budur. Ksilan 311% ile en bağıl absorbans gösterdi. Bunun nedeni daha baskın furanoz forma ksiloz ulaşılan yüksek molar tükenme katsayısı. 0.1 g / L glukoz seviyesinde miktar sınırı ulaşıldı, hem de bir konsantrasyonda &lt;0.05 g / L&#39;de tespit limiti. Bununla birlikte, tarama pozitif suşları için tespit limiti daha yüksek 0.7 g / L ve bu nedenle, tahlil, iyi bir performans göstermiştir. güvenilir sonuçlar elde etmek için, jel-filtreleme sonra kalan glikozu, bir glikoz-tahlili ile belirlendi ve bu değer, fenol-sülfürik asit yönteminden değerinden çıkarıldı. Otomatik glukoz-tahlil seyreltme: performansekimi (sulandırma 1: 100), daha sonra glukoz belirlenmesi araştırılmıştır. Bunun için, süpernatan 10 ul aspire ve seyreltme 180 ul dağıtma on kat ile derin oyuklu bir plaka içerisinde GKD 2 O 990 ul transfer edildi ve karıştırıldı. İkinci önemli bir adım jel filtrasyon sonrası glikoz tahlili 1:10 seyreltme için yalnızca 5 ul alikot doğru pipetleme idi. GKD 2 O seyreltme 25 ul oluşturmak için ilk 50 ul-ucu ile transfer edilmiştir, GKD 2 O ve 5 ul jel süzüntünün daha sonra 20 | il bir araya aspire edildi. Bu ucu dışarı 5 ul alikotununLH daha iyi uzaklaştırılmasını sağlar. Her ikisi de seyreltme adımları bir glikoz-tahlil yoluyla çeşitli glukoz standartları ile doğrulandı. İki örnek konsantrasyonları için sonuçları Tablo 6&#39;da verilmiştir: 1. 100 seyreltme glukoz içeriğinin belirlenmesi için ekimi bir CV &amp; # ile hem standartları yüksek hassasiyet gösterdi sonra60,% 1.2. Aynı zamanda, daha yüksek standartta için doğruluk 11.6 (% hatası) kadar oldu. glukoz belirlenmesi kültivasyonu takiben geriye kalan glukoz içeriği temsil eder ve bu nedenle, polimer, algılama için önemli değildir Ancak, bu ihmal edilebilir düzeydedir. Jel süzme sonra kalan glikoz 1:10 seyreltme CV &lt;% 1,4 ve doğrulukla &lt;% 2.1 hatası çok güvenilir sonuçlar göstermiştir. buharlaşma göz önünde: tarama, ilk glukoz-tahlil ilk adım 3.5 saat gerektirir. Bu zaman çerçevesi kapatılmamış MTP depolama etkisinin olup olmadığını öğrenmek için, glukoz-tahlil kalibrasyon standartları 50 ul ve robot atlıkarınca 3,5 saat kapaksız saklandı. kalibrasyon aralığı (45-4,5 mg / L), numune konsantrasyonu hemen hemen artmıştır. Bir artış - buharlaşma nedeniyle -% 2,1 altında ve sadece iki düşük konsantrasyonlarda (1.8 ve 0.9 mg / L)% 12.4 kadar ulaşmıştır (oldu <strong&gt; Tablo 7). Jel filtrasyon: non-metabolize glikoz Yüksek miktarda hidrolize polimerden glikoz kantitatif tespiti rahatsız. Bu nedenle, bir jel filtrasyon aşaması, kültivasyondan sonra, kalan glukoz kaldırmak için gerekli oldu. Buna ek olarak, jel filtrasyon monomer analizinde analiz arka en aza indirmek için, glikoz daha tuzları ve monomerik karbohidrat bileşikler diğerlerinden süpernatan ihtiva eden polimer temizler. Jel filtrasyon adımında filtre 35 ul iyi merkezine yerleştirildi. otomatik sistemi içerisindeki jel-filtrasyon sağlamlığı doğrulama için, 9 g / L glikoz kadar 0.045 sekiz kalibrasyon standartları süzüldü (n = 8). Her konsantrasyon glükoz, her zaman ilk değer (Tablo 8)% 95&#39;den daha fazla azalmıştır. Bu şekilde, jel filtrasyon glikoz çeşitli konsantrasyonlarda çok iyi sonuçlar elde edilmiştir. Buna ek olarak, diğer glukoz jel f sonra, filtrasyon da glukoz-testi ile belirlenmiştir ve hidrolize polimer için glukoz eşdeğeri doğru miktarda almak için fenol-sülfürik asit belirlenmesi çıkarıldı. Piruvat-deney: Her şeyden önce, bu hidroliz aşamasından nötralize edildi ve seyreltildi (1:10), TFA-matris, enzimatik reaksiyonu kesintiye uğrattığı araştırılmıştır. Bu nedenle, tam tahlil ile bir kez ve bir kez matris olmadan, iki kez gerçekleştirilen ve güvenilir sonuçlar gösterdi. Son olarak, 16 ticari olarak temin edilebilen polimerlerin piruvat içeriği başarıyla ölçülmüş ve Şekil 3&#39;te gösterilmektedir. Genel olarak, bu 16 polimerler arasından, sadece süksinoglikan ve ksantan doğal piruvat içeren bilinmektedir. Bizim piruvat-tahlil ile bu polimerlerin iki doğru bir şekilde tespit edilmiştir. skleroglukan olarak, velan sakızı ve ksilan piruvat da önemli miktarda tespit edilmiştir. Genel olarak, yaklaşım yeteneği onaylanmış ve piruvat-tahlil s olduyüksek performans howed. Bu hidroliz sonrası farklı polimerler içinde piruvat tespit etmek mümkün olduğu kanıtlanmıştır. Karbonhidrat parmak izi: Otomatik tarama tüm analitik modülleri gerçekleştirdikten sonra, potansiyel EPS üreticileri karbonhidrat parmak izi analizi için seçildi. Bunun için, çeşitli kriterler uygulanmıştır: viskozite 1) pozitif bir gözlem santrifüj sonra ve / veya süzme sonrasında. 2) Yağış süzme öncesinde ve sonrasında. Gözlenen lifler ve yongalar olumlu olarak değerlendirilmiştir. fenol-sülfürik asit yöntemiyle 3) Glikoz eşdeğer değer. &gt; 700 mg / L değerleri arasında pozitif olarak değerlendirilmiştir ve 300 ve 700 mg / L arasındaki değerler varsayılan EPS üreticileri olarak değerlendirilmiştir. iki ya da üç kriter pozitif olarak değerlendirilmesi durumunda, suşları daha karbohidrat parmak izi analizi için seçildi. Kriterler EPS tarama (örneğin, düşük viskoziteli EPS) bireysel amaca yönelik özelleştirilebilir. Bizim yaklaşım effici bulma amaçlıent üreticileri EPS. Sadece EPS küçük miktarlarda üretmek suşları ararken glikoz eşdeğeri değerlendirme sınırı azaltılmalıdır. Teknik yarar ve gelecek uygulamalar: Bu protokolün ilginç bir özelliği adımlar ve farklı analitik modüllerin modüler karakteridir. Bunlar bireysel gereksinimlere göre ayarlanabilir, farklı şekillerde kombine edilebilir ve yeni modüller kolayca uygulanabilir. Ayrıca, analiz modülü ayrı ayrı kullanılabilir örneğin HT-PMP-türetme modülü ile bir arada hidroliz modülü 96 oyuklu bir formatta, farklı polimer çözeltileri (1 gr / lt) bir monomerik bileşimin analizi gerçekleştirebilir. tam tarama pipetleme şemasında herhangi bir değişiklik olmadan manuel olarak ele alınabilir bir sıvı taşıma sistemine erişimi kalmadan laboratuvarlar için. Ancak, sıvı taşıma sistemi kullanılarak en fazla 768 suşları (yerine Screene eğer 192 suşları verimi artırırgün başına) elle d. Burada açıklanan protokol farklı cins için bir tarama özelliğine sahiptir ve bu nedenle, yeni bir EPS üreticileri belirlemek ve bir yaklaşımı (Şekil 4) da karbohidrat parmak izi analizi büyük Strain collections&#39;da taranması için kullanılabilir. Ayrıca, fukoz gibi nadir şekerler, üronik asitler ya da bilinmeyen bir şeker içeren polisakaridler için hedeflenen bir tarama detaylı bir monosakarit analizi ile yapılabilir. Ayrıca, tanımlanan oranlarda çeşitli şeker kombinasyonları tespit edilebilir. Bu zaten bilinen EPS veya yeni EPS yapısal olarak ilişkili türevleri basit tanımlanmasını sağlar.

Mikrobiyal eksopolisakaridlerin (EPS), çeşitli biyolojik fonksiyonları yerine getirir bir polimer yapısal yüksek gruptur. Genellikle, farklı monomer tipleri (şeker, şeker türevleri, şeker asitleri), bu monomerlerin ve ikameleri arasındaki bağlar ile ayırt edilir kompleksi tekrar birimlerinin, örülmüştür. Mikrobiyal polisakkaritler yapısal çeşitlilik gıda 1 gibi farklı alanlardaki uygulama sağlayan bu molekül sınıfına, kozmetik 2,3, inşaat kimya 4 veya su arıtma 5 üyelerine oldukça farklı özelliklere bahşeder. ayrıca yeni, doğal mikrobik polisakaritler tanımlanması ve yapısal varyantları tekniği umut vadeden yaklaşımlar temsil bu biyolojik bazlı ve sürdürülebilir polimerlerin uygulama alanını genişletmek için. Her iki durumda da, hızlı bir tarama yöntemidir hızla thei için mikrobiyal suşların geniş bir sayıda tarama için gereklidirr polisakkarit oluşumu ve ürünlerini analiz etmek. Bu nedenle, en son monomer bileşimin 6 belirlenmesini içeren, doğal izolatları ya da genetik mühendisliği suşu türevleri mikrobiyal polisakarit üretimi analizi için 96-çukurlu HT-görüntüleme platformunu geliştirdik. ~ 500 doğal izolatların bir ilk tarama turu için bu platformu uygulamak bize EPS üretmek mümkün varlık olarak yalnızca yaklaşık 10 İzole edilen suşların% 20 tespit izin (veriler gösterilmemiştir). Bu analiz suşların% 80-90 uygulanan koşullar altında EPS vermedi ve bu nedenle ayrıntılı bir karbohidrat parmak izi bir başka analizi, gerekli olmadığı anlamına gelmektedir. monomerik kompozisyon bu son derece sofistike kimlik olarak özellikle veri analizi için, hızlı bir ön tarama yöntemi, EPS üretimi olumlu suşları belirlemek için büyük ölçüde verimliliği artıracak, zaman alıcı bir süreçtir. Ayrıca, reaktifler,UHPLC-UV-ESI-MS de sarf malzemeleri ve ölçüm süresi azalacaktır. Bir yandan yöntem son derece güvenilir yaparken Buna ek olarak, farklı analitik modülleri paralel en az iki 96-yuvalı plakalar kılavuzu taşıma komplike Diğer taraftan, ve bu şekilde yöntemin tam potansiyelini sınırlamaktadır. Bu nedenlerden dolayı, biz otomatik tarama platformu geliştirmeye karar verdi. Bu nedenle, absorbans ölçümü dayalı olarak, toplam şeker içeriği tam otomatik hızlı bir algılama yöntemi mevcut karbohidrat parmak tekniğin modüler bir biçimde bir araya. Fenol-sülfürik asit yöntemi hala bakteri ve bitki polisakkaritler 7,8 toplam karbonhidrat içeriğinin hızlı tayini için tercih yöntemi temsil eder. Bu yöntem, ilk 96-yuvalı plakalar 10,11 daha küçük ölçekli, farklı uygulamalar ve örnek boyutları için Dubois ve diğ. 9 ile tarif uyarlanmıştır. phenol-sülfürik asit yöntemi tüm monomerik, oligomerik ve örnekleri polimerik karbonhidrat summating, tek bir değere göre, toplam karbonhidrat içeriği ölçer. dikkate bu yönleri alarak, uygun bir yetiştirme ortamının seçimi, bu yöntemin uygulanması için çok önemlidir. oligomerik veya maya özü gibi polimerik karbohidrat bileşikleri ihtiva eden kompleks ortam değişmiş bir polimer içeriğine yol açabilir ve bu nedenle, kesinlikle kaçınılması gerekmektedir. Bundan başka, şekerler, yüksek miktarlarda soylarının yetiştirilmesi için C-kaynağı olarak kullanılır. yetiştirme sürecinden kalan karbonhidratların yüksek seviyelerde olumsuz EPS içeriği ölçümü etkileyebilir. Bu nedenle, tanımlanmış ve saf şekerler kullanılması tavsiye edilir. Deneylerimizde hücrelerinin kültivasyonu için glükoz kullanılır. yetiştirme sonrası kalan glukoz jel filtrasyon yoluyla indirgendi ve glikoz tahlili ile belirlendi. Son olarak, glukoz eşdeğeripolisakaritlerin s fenol-sülfürik asit yöntem ile tespit edilmiş toplam karbonhidrat içeriği jel filtreleme işleminden sonra kalan glukoz çıkarılarak hesaplanmıştır. fenol-sülfürik asit metodu ile kombinasyon halindeki jel filtre ve glukoz-deney güvenilir sonuçlar sağlar ve ilk, tamamen otomatik kontrol sistemi olma yeteneğine sahiptir. çökeltme ve viskozitedeki bir artış gözlenmesi: İki yeni analitik modül EPS algılama sistemi bilgi miktarını artırmak için otomatik tarama platformu alındı. Birçok farklı EPS - örneğin suksinoglikanlar, ksantan ve kolon asit 12 - şeker pozisyonları C4 ve C6 olmayan bir karbonhidrat piruvat ketal ile modifiye olduğu bildirilmektedir. (Sadece süksinil yarım esterleri ve üronik asitler gibi) bu piruvat ketaller fiziksel prope için, bu nedenle polianyonik doğaya katkıda bulunmak veiki değerlikli katyon köprüleri 13 vasıtasıyla etkileşim polimerin ilgili taraflar. bu özel polimerler belirlemek amacıyla piruvat belirlenmesi ek başka bir analiz modülü olarak kurulmuştur. Bu polisakkarit ikamesi ve potansiyel makroskopik özellikleri hakkında bilgi arttırır. tüm modülleri birleştiren farklı EPS belirlenmesini yanı sıra EPS üreticilerinin hızlı ve etkin belirlenmesini sağlar. Bu yaklaşımla tarama platformu iki ana bölümden (Şekil 1) bölünebilir. Otomatik tarama (Bölüm I) iş akışı tam otomatik oluşur içinde (Tablo 1) hızla üreten suşlar EPS preselect için. karbonhidrat parmak izi analizi (Bölüm II) nicel önceden seçilmiş suşları tarafından üretilen EPS monomer bileşimi belirler. Böylece, veri analizi büyük gerilme koleksiyonları tarama optimize etmek için minimize edilmiştir. Butek otomatik tarama vadede ve günde 768 suşları günde mümkün olan iki çalışan ile 384 suşları analiz imkanı sunuyor. Ayrıca, karbonhidrat parmak izi analizi, tüm belirlenen EPS daha da ayrıntılı bir bakış sunuyor. Bu yönelik analiz ve EPS önce tarif kimyasal yapıları ile karşılaştırıldığında sadece biraz farklı EPS varyantları ya da tamamen yeni olanlar tanımlanmasını sağlar.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="126">
			<td height="126" style="height:126px;width:335px;">96 well deep well plate 2.0 mL (DWP)</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">780271</td>
			<td style="width:224px;">Main-culture (CP 1-1 to 1-4), equilibration plates for gel-filtration (CP 1-6, 1-7, 2-6 and 2-7), 1:100 dilution for the&#160; glucose assay (CP 4-1 to 4-4) containing 990&#181;l of ddH2O</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Breathable Sealing Film</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">BF-400-S</td>
			<td style="width:224px;">Incubation film for pre- and main-culture DWP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Aluminum Sealing Film</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">PCR-AS-200</td>
			<td style="width:224px;">-80 &#176;C storage film for glycerol-stock plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">MCA96 Nested Disposable Tips 200 &#181;l</td>
			<td style="width:185px;">TECAN</td>
			<td style="width:143px;">30038619</td>
			<td style="width:224px;">Worktable position (WTP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">A/B glass-filter-plate 1&#181;m</td>
			<td style="width:185px;">Pall Corporation</td>
			<td style="width:143px;">PN 8031</td>
			<td style="width:224px;">Stored on collector plate (CP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well micro titer plate V-Bottom</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">651201</td>
			<td style="width:224px;">Collector plate for filtration plate (CP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well SpinColumn G-25</td>
			<td style="width:185px;">Harvard Apparatus</td>
			<td style="width:143px;">74-5612</td>
			<td style="width:224px;">Stored on washing DWP (CP 1-6, 1-7, 2-6 and 2-7)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well micro titer plate V-Bottom</td>
			<td style="width:185px;">Nunc</td>
			<td style="width:143px;">249944</td>
			<td style="width:224px;">Collector plate for gel-filtration plate (CP 3-1 to 3-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Nested Disposable Tips SBS 50 &#181;l tips</td>
			<td style="width:185px;">TECAN</td>
			<td style="width:143px;">30038609</td>
			<td style="width:224px;">Carousel position (CP 4-6, 4-7 and 5-1 to 5-6)</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:335px;">Trough 250 ml</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">Res-SW96-HP</td>
			<td style="width:224px;">Water WTP 1-1, Glucose assay reagent-mix WPT 1-2, ammonium-acetat buffer pH 5.6 (CP 5-7)</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:335px;">96-well micro titer plate F-Bottom (MTP)</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">655101</td>
			<td style="width:224px;">precipitation 1 (CP 6-1 to 6-4), pH-value (CP 7-1 to 7-4), precipitation 2 (CP 8-1 to 8-4), phenol-sulfuric-acid method (CP 8-5 to 8-8), glucose-assay (CP 9-1 to 9-9), dummy plates (WTP 3-1, 3-2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">96-well silicon cap mat&#160;</td>
			<td style="width:185px;">Whatmann</td>
			<td style="width:143px;">7704-0105</td>
			<td style="width:224px;">Cover mat for MTP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">200 &#181;l pipette tips</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:143px;">70.760.002</td>
			<td style="width:224px;">For manually handling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">1000 &#181;l pipette tips</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:143px;">70.762</td>
			<td style="width:224px;">For manually handling</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well-PCR micro titer plate&#160;</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">781350</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatisation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">TPE (thermoplastic elastomer) cap mat&#160;</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">781405</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatisation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:335px;">Filter plate 0.2&#160;&#181;m Supor,&#160;</td>
			<td style="width:185px;">Pall Corporation</td>
			<td style="width:143px;">PN 8019</td>
			<td style="width:224px;">Filtration of samples for UHPLC-ESI-MS analysis with a MTP collector plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Pipette Tips LHS 5-300 &#181;l</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">732150</td>
			<td style="width:224px;">Brand LHS system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">ABTS (2.2-azino&#8209;bis-(3&#8209;ethylbenzthiazoline)-6-sulfonic&#160;acid)</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">A1888</td>
			<td style="width:224px;">Glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Glucose oxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">G2133</td>
			<td style="width:224px;">Glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Horseradish peroxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6782</td>
			<td style="width:224px;">Glucose-assay, pyruvat-assay</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:335px;">DA-64 (N-(Carboxymethylaminocarbonyl)-4.4&#39;-bis(dimethylamino)-diphenylamine sodium salt)</td>
			<td style="width:185px;">Wako Chemicals GmbH</td>
			<td style="width:143px;">043-22351</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Pyruvate oxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4591</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Potassium phosphate dibasic</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">P749.3</td>
			<td style="width:224px;">Pyruvat- and glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Potassium phosphate monobasic</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">3904.3</td>
			<td style="width:224px;">Pyruvat- and glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Thiamine pyrophosphate</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">C8754</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Magnesium chloride hexahydrate</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">31413</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">2-Propanol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20922.466</td>
			<td style="width:224px;">Precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Phenol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20599.231</td>
			<td style="width:224px;">Phenol-sulfuric-acid method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Sulfuric acid</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">4623.4</td>
			<td style="width:224px;">Phenol-sulfuric-acid method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Trifluoroacetic acid</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">T6508</td>
			<td style="width:224px;">Hydrolysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Ammonium solution</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">P093.1</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Ethanol absolut</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20821.321</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Phenol red&#160;</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:143px;">B21710</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">1-Phenyl-3-methyl-5-pyrazolone</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">M70800</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Methanol LC-MS</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">83638.320</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Acetonitril LC-MS</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">83040.320</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Acetic acid</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">338826</td>
			<td style="width:224px;">PMP-derivatization,</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Ethanol absolut</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20821.321</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Methyl red&#160;</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:143px;">36667</td>
			<td style="width:224px;">pH-value</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Robotic liquid handling system&#160;</td>
			<td>Tecan</td>
			<td>Freedom EVO</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid handling station LHS</td>
			<td>Brand</td>
			<td>709400</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tip-Adapter&#160;</td>
			<td>Brand</td>
			<td>709434</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Ends MC 20-300&#181;L</td>
			<td>Brand</td>
			<td>709416</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adapter 60mm</td>
			<td>Brand</td>
			<td>709430</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
	</tbody>
</table>

automated modular high throughput exopolysaccharide screening platform coupled with highly sensitive carbohydrate fingerprint analysis

Birçok mikroorganizma üreten ve tıp alanları, gıda uygulamalarında ya petro-bazlı kimyasallar yerine önemli etkileri eksopolisakaridleri (EPS), salgılayan yeteneğine sahiptir. Biz türü ve mikroorganizmalar tarafından üretilen EPS miktarının hızlı ve güvenilir analiz sağlayan bir sıvı taşıma sistemi otomatik hale analitik platformu açıklar. Bu kullanıcı, yeni doğal mikrobiyal ekzopolisakkarit üreticileri tespit etmek ve yüksek verim (HT) bir gün içinde karşılık gelen polimerlerin karbohidrat parmak izi analizi sağlar. Bu platformu kullanarak, mühendislik yaklaşımlarına elde edilebilir gerilme koleksiyonları yanı sıra gerilme varyantları kütüphaneleri taranabilir. Platform iki ana bölüme protokolünün bir ayrılık sağlayan modüler kurulum vardır. İlk olarak, farklı bir polisakarit algılama modülleri birleştiren otomatik bir tarama sistemi vardır: Viscosit bir yarı-kantitatif analizibir santrifüjleme adımı alkol çökeltme yoluyla, polimer oluşumu, bir analiz ve bir fenol-sülfürik asit dönüştürme aracılığıyla toplam karbonhidrat içeriğinin belirlenmesi yoluyla Y oluşumu. Burada, dönemde başına 384 suşları kadar taranması mümkündür. İkinci bölüm, iki temel modülleri birleştirerek ilk bölümünde tanımlanmış tüm seçili EPS üreticileri için ayrıntılı bir monosakarit analizi sağlar: UV ve elektrosprey iyonizasyon iyon tuzağı algılama (birleştiğinde ultra yüksek performanslı sıvı kromatografisi ile tam bir monomer bileşiminin analizi UHPLC UV-ESI-MS) ile bir renk oluşumu ile birleştirilir enzimatik oksidasyon aracılığıyla, bir polimer ikame (piruvat ketal varlığı) ve piruvat belirlenmesi. Bu tarama platformun tüm analitik modüllerinin farklı şekillerde bir araya getirilmiş ve kişisel gereksinimlere göre ayarlanabilir. Buna ek olarak, hepsi elle hareket ettirilmesi veya bir sıvı taşıma sistemi ile gerçekleştirilebilir. Böylece, eleme plaTForm çeşitli EPS tanımlamak için büyük bir esneklik sağlar.

Watch this Scientific Journal Video about Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis at JoVE.com

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Otomatik Modüler Yüksek Verimli Ekzopolisakkarit Tarama Platformu Oldukça hassas Karbonhidrat Parmak İzi Analizi ile birleştiğinde

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

automated-modular-high-throughput-exopolysaccharide-screening

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11.4K Görüntüleme.  Technische Universität München. We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Video: Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Mitokondri Yüksek Coupled Sıçan Kalp hazırlanması

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Floresan ile yüksek verim Tarama ve Biyoalgılayıcı C. elegans Suşlarının

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Mantar Endoglucanase Faaliyet Yüksek Verimli Tarama Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Bir ev yapımı Çift kızdırma Lusıferaz Testi kullanarak yüksek verim Fonksiyonel Taraması

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Yüksek Verim Dizilimine tarafından MicroRNA ölçümü için Küçük RNA molekülleri, yüksek Verimli ligasyonu

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Yüksek Verimli Tarama kullanma Kinaz-substrat Çiftlerinin Kimlik


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

Süspansiyon HEK293 hücrelerinin kısa süreli transfection ile Rekombinant İnsan Proteinlerinin Yüksek Verimli İfadesi

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Çok enzim Yüksek verim Genetik Enzim Eleme Sisteminin Kullanılması Eleme

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...