Biz mükemmel teknik servis ve John Ralph, University of Wisconsin için değerli tavsiyeler, tartışmalar ve kavak ahşap örnek Matthew Robert Weatherhead için minnettarız. Bu çalışma, Kimya Bilimleri, Yerbilimleri ve Biosciences Bölümü, Temel Enerji Bilimler, Fen Dairesi, ABD Enerji Bakanlığı (ödül hayır. DE-FG02-91ER20021) ve ABD Enerji Departmanı (DOE) tarafından Büyük Göller Dairesi tarafından finanse edildi Biyoenerji Araştırma Merkezi (DOE BER Office of Science DE-FC02-07ER64494).

1. Hücre duvar izolasyonu <ol><li> eziyet kabaca 60-70mg hava veya retschmill (1 dak, 25 Hz) kullanarak bir 2ml Sarstedt vidalı kapaklı tüp 5.5 mm paslanmaz çelik bilya ile kurutulmuş bitki materyali dondurmak. Bir alternatif, yüksek verimli bir taşlama ve robot olarak adlandırılan iWall dağıtım Part I 3 açıklanmıştır . </li><li> hücre duvarı izolasyon prosedürü ile devam etmeden önce çelik topları kaldırmak Hücre duvar malzemesi hazırlanması ayrıntılı protokol Part I 3 gösterilir. Protokol yazılı adımları burada tamlığı için. </li><li> 1.5 ml,% 70 sulu etanol ve iyice vorteks eklemek </li><li> pelet için 10 dakika alkol çözünmeyen kalıntı 10.000 rpm&#39;de santrifüj </li><li> süpernatantı süzün veya aspirat </li><li> kalıntı kloroform / metanol (1:1 v / v) solüsyonu 1.5 ml ekleyin ve iyice tüp sallamak pelet tekrar süspansiyon </li><li> 10 dakika 10.000 rpm&#39;de santrifüj ve supernatant süzün veya aspirat </li><li> aseton 500 ul tekrar süspansiyon pelet </li><li> 35 yaşında bir hava akışı ile solvent buharlaşması ° C kuruyana kadar Gerekli kurutulmuş örnekleri daha fazla işlem kadar oda sıcaklığında saklanabilir. </li><li> Örnek 1,5 ml 0,1 M sodiumacetate tampon pH 5.0 yeniden askıya pelet nişasta kaldırılması başlatmak için. </li><li> 20 dakika Sarstedt boru ve ısı kap. 80 ° C ısıtma bloğu. </li><li> buz üzerinde süspansiyon serin </li><li> pelet için aşağıdaki maddeler eklemek: 35 0.01% Sodiumazide ul (NaN 3), 35 ul Amilaz (50 μg/1mL H 2 O; Bacillus türleri, SIGMA), 17 ul Pullulanase (18.7 adet basil acidopullulyticus; SIGMA) . Ve iyice vorteks tüp kap. </li><li> Süspansiyon 37 gece boyunca inkübe ° C çalkalayıcı. Tüpler yönlendirmek yatay yardımcıları karışım geliştirdi. </li><li> ısı süspansiyon 100 ° C sindirim sonlandırmak için bir ısıtma bloğu 10 dk. </li><li> santrifüj (10.000 rpm, 10 dakika) ve atın supernatant içeren çözünür nişasta </li><li> 1.5 ml su ekleyerek karıştırın, centriguation ve çamaşır suyu şişeden kalan pelet üç kez yıkayın. </li><li> aseton 500 ul tekrar süspansiyon pelet </li><li> kuruyana kadar hava akımı 35 ° C ile solvent buharlaşır. Daha iyi kurutma için bir spatula ile tüp içindeki malzemeyi kırmak için de gerekli olabilir. Kurumuş malzeme izole hücre duvarı (lignocellulosics) sunar. Gerekli kurutulmuş örnekleri daha fazla işlem kadar oda sıcaklığında saklanabilir. </li></ol> 2. Matrix Polisakarid kompozisyon Bu yöntem, özünde Albersheim 1 tarafından yayımlanan yöntemi bir değişiklik. <ol><li> Duvar malzemesi monosakkarit kompozisyonunu belirlemek için, elle veya iWall, bir robot taşlama ve tartım robotu kullanarak highthroughput moda ya 2ml starstedt tüpler içine hücre duvar malzemesi 2 mg ağırlığındadır. </li><li> , Bir internal standart olarak bir İnositol solüsyonu (5mg/ml) 20 uL ekleyin. 2 mg hücre duvarı örnek için 100 ug eklemek için önerilir. </li><li> tüpün alt hücre duvarı malzeme toplamak için tüp duvarları 250 ul aseton ile temizleyin, durulayın ve aseton çok hafif hava akımı altında buharlaşması. </li><li> Zayıf asit hidrolizi için her numune 2M trifloroasetik asit (TFA) 250 ul ekleyin. Hiçbir malzeme tüp duvarlarına sıçrayan sağlamak için dikkatle TFA ekleyin. </li><li> sıkı kapağı ve 121 90 dakika boyunca inkübe ° C ısıtma bloğu. </li><li> ısıtma blokları ve örnekler buz üzerinde soğutun. </li><li> Tüpleri 10 dakika 10.000 rpm&#39;de santrifüj. </li><li> pelet malzeme rahatsız değil emin olun bir bardak vidalı kapaklı şişeleri monosakkarit elde edilen matris polisakkarit içeren asidik süpernatant 100 ul aktarın. Pelet (3) aşağıdaki kristalin selüloz tayini için kullanılan </li><li> hafif bir hava akımı altında bir buharlaştırma cihazı, cam tüp içinde TFA buharlaşır. </li><li> 300 ul 2-Propanol, girdap ve buharlaşması 25 ° C (toplam üç kez tekrar) </li><li> Alditol asetat türevlendirme prosedürün ilk adımı, bunlara karşılık gelen alditols Monosakkaritler bir azalma yapmaktır. Bu amaçla her kurutulmuş örnek bir çözüm sodyum bor hidrür, 200 ul ekleyin. Taze bir çözüm her zaman başına 1M Amonyum hidroksit 1 ml sodyum bor hidrür 10mg kullanarak hazırlayın. </li><li> Cam şişe, 1,5 saat boyunca oda sıcaklığında terk </li><li> 150 ul buzul asetik asit ekleyerek çözüm nötralize </li><li> 25 girdap ve buharlaşması ° C. </li><li> 250 ul asetik asit / Metanol (01:09 v / v), girdap ve 25 buharlaşmaya ˚ C </li><li> (toplam üç kez tekrar) 250 ul Metanol ekleyin ve hava akışı altında buharlaşması </li><li> Alditols asetilasyonu için asetik anhidrit, 50 ul ve Pyrid 50 ul ekleyin20 dakika için 121 ine, girdap ve inkübe ° C ısıtma bloğu. </li><li> buz bloğu serin numuneler oda sıcaklığında yaklaşık için geçici azalma beklerken. </li><li> reaktifler oda sıcaklığında hafif bir hava akımı altında buharlaşır. Dikkatli olun: alditol asetatlar havai. </li><li> hava altında 200 ul Toluen ekleyin ve buharlaşması (x3) </li><li> Son adımları alditol asetatlar ayıklanır. İlk olarak, etil asetat ve hafifçe girdap 500 ul ekleyin. </li><li> 2 ml su, şapka tüpleri ve vorteks ekleyin. </li><li> net ayrı katmanlarda (etil asetat, üst alt su) elde etmek için 5 dakika için 2,000 RPM tüpler santrifüj </li><li> GC / MS ekler ile şişeleri içine etil asetat tabaka pipet 50 ul. </li><li> aseton 100 ul GC-şişe ve kapak ekleyerek sulandırmak. Örnek konsantrasyonu çok yüksek ise, örnek hacmi ve seyreltme hacim GC / MS aşırı yüklenmesini önlemek için ayarlanabilir. GC-flakon, 4 ° C, GC / MS analizi hemen devam gelmez eğer saklanabilir </li><li> Numuneleri, bir quadrupole MS ile donatılmış bir GC enjekte ama alev iyonizasyon dedektörü de uygundur. Supelco SP-2380 (30mm X 0.25mm x 0.25 mikron film kalınlığı) sütun 4min çözücü bir gecikme ve 1.5ml/min bir akış hızı ile kullanılır. Enjekte örnekleri aşağıdaki sıcaklık programına tabi tutulur: 160 İlk tutun ° C için 2 dk, 20 ° ile 200 C / dak rampa ° C ve 5 dakika boyunca tutun; 20 ° ile 245 C / dak rampa ° C ve basılı tutun 12 dakika; başak 270 ° C ve 160 ° C 2,26 ilk sıcaklık soğutma önce 5 dakika boyunca basılı tutun) Peaks kitle profilleri standartlar ve / veya alıkonma süreleriyle tanımlanır. Monosakkaritler standart eğrileri dayalı sayısal vardır. </li></ol> 3. Kristal Selüloz İçerik Bu yöntem aslında Updegraf 8 tarafından açıklanmıştır. İzole hücre duvar malzemesi (1) veya zaten 2M TFA (2.8 &#39;e bakınız) ya kalan pelet asit tedavisi hemen sonra (2.8) veya TFA ile tedavi edildikten duvar malzemesi: Bu prosedür için başlangıç ​​materyali sayısı vardır pelet, 2-propanol ve kuru ile yıkanır olmuştur. <ol><li> Updegraff reaktif vidalı kapaklı cam tüp 1 ml (nitrik asit: su, 08:01:02 v / v asetik asit) TFA pelet ekleyin. </li><li> Kapağını sıkıca kapatın tüp 100 ısıtma bloğu, girdap ve ısı ° C de 30 dakika. Bu tedavinin bir sonucu olarak sadece kristal selüloz pelet çözünmez kalır. </li><li> Blok, oda sıcaklığında veya soğuk buz üzerinde soğutun örnekleri </li><li> 15 dakika 10.000 rpm&#39;de santrifüj örnekleri </li><li> Pelet ve kaldırılır rahatsız pelet hiçbir malzeme olmadığı sağlamak süpernatant atın. Bu amaçla yaklaşık bırakın. 150 tüp süpernatant ul. </li><li> santrifüj sallamak, 1.5 ml su ekleyin ve yukarıda yaptığımız gibi süpernatant atın. </li><li> 1.5 ml aseton kullanarak işlem 3 kez yıkama tekrar </li><li> Hava kuru pelet hava ile çok nazik ya da geceleme bankta kurumaya bırakın </li><li> pelet (kristal selüloz) Saeman hidroliz ne denir artık tamamen glikoza hidrolize olur. Bu amaçla Sarstedt tüp 175 ul% 72 Sülfürik asit eklemek için </li><li> başka bir 15 dakika için 30 dakika, girdap ve inkübe oda sıcaklığında inkübe </li><li> 825 ul su ve vorteks eklemek </li><li> 5 dakika 10.000 rpm&#39;de örnekleri santrifüj. Tüp içinde kalan bazı kahverengi çözünmez malzeme, lignin, olabilir. </li><li> Süpernatantı glikoz içeriği kolorimetrik anthrone assay kullanılarak denenmiştir. Bu testte 96 polistiren mikrotiter plate yapılır. </li><li> Standart eğri 1mg/ml şekeri stoku (0 ° C&#39;de depolanan) ve 0, 2, 4, 6, 8 ve 10 ul pipetleme, 0, 2, 4, 6, 8, ve 10 ug standartları kopyasını oluşturmak için ayrı uygun bir kuyuya. Her iyi 100 ul su ile doldurun. </li><li> ayrı hücrelere ancak standart olarak aynı mikrotiter plate ul her örnek süpernatant 10 ve 90 ml su ekleyin. </li><li> taze hazırlanmış Anthrone Reaktif (Anthrone konsantre sülfürik asit, 2 mg anthrone / ml sülfürik asit içinde çözünmüş), 200 ul eklemek </li><li> 80 az 30 dakika ısı plakası ° C fırında (alüminyum ısı yayıcı). Glikoz içeren örnekler, sarı, mavi-yeşil renge çevirin. </li><li> plaka oda sıcaklığına kadar soğumasını bekleyin ve iyice çalkalayın. </li><li> mikrotiter plate okuyucusu kullanarak 625 mm plaka emilimini okuyun. </li><li> Glikoz (ve dolayısıyla kristal selüloz içeriği) absorbans dayalı, aynı plaka üzerine kurulmuş standart eğri göre hesaplanır. </li></ol> 4. Temsilcisi Sonuçlar Bir duvar analizi bir örnek Şekil 2&#39;de sunulmuştur. Bu durumda kavak kök (ağaç) protokol bölümünde belirtilen çeşitli işlemler analiz edildi. Matris polysaccharide yapısı, bitki hücre duvarları tipik şeker mevcut tanımlayan bir örnek kromatogram vurgulanır, fucose, ramnoz, ksiloz, arabinoz, galaktoz, mannoz ve glikoz (ve iç standart inositol). Yüksek Xylose içeriği tarafından gösterildiği gibi, kavak başlıca hemicellulosic bileşeni Xylan. Ancak, bu şekerlerin bol miktarda hammadde used4 bağlı olarak değişecektir. Bu analizde glikoz ve amorf selüloz hemiselüloz xyloglucan türetilmiştir. Veri analizi sayesinde mol% veya ug / mg duvar malzemesi (veya kuru ağırlık) olarak sunulabilir. Kristalin selüloz içeriği kendi kendini açıklayıcı, bir duvar kuru ağırlığının% 20-50 arasında değerler bekleyebilirsiniz. Bölüm I3 ve burada sunulan sonuçlar dayanarak kavak ağacından lignocellulosic kompozisyonu% 21 lignin,% 30 hemiselüloz ve% 41 kristal selüloz. Geri kalan kül olacaktı. <img src="/files/ftp_upload/1837/1837fig1.jpg" alt="Şekil 1" /> Şekil 1: lignocellulosic analiz Genel Bakış Hücre duvarları (lignocellulosics) ham kurutulmuş bitki materyali izole edilmiştir. Duvar malzemesi, sonra alikotları içine ağırlıklı ve çeşitli deneyleri için bölünmüştür. Matrix polisakkarit kompozisyon duvar malzemesi, zayıf bir asit (2M TFA) ile tedavi alditol asetatlar elde edilen çözünür Monosakkaritler derivatizing, ve GC-MS analizi sonra kurulmuştur. Zayıf asit tedavisi kalıntı sözde Updegraff reaktif geride bırakarak sadece çözünmez crystlline selüloz ile yıkanır. Selüloz sülfürik asit çözünür ve glikoz içeriği belirleyen bir kolorimetrik yöntemi ile sayısal. Buna paralel olarak, Bölüm I 3 açıklandığı gibi lignin içeriği ve bileşimi tespit edilebilir. <img src="/files/ftp_upload/1837/1837fig2.jpg" alt="Şekil 2" /> Şekil 2: kavak (Populus tremoloides) kavak ahşap Kapsamlı lignocellulosic analiz Ahşap cips açıklanan protokole tabi tutuldu. Sol üst: Matrix polisakkarit bileşimi; FUC fucose; Rha ramnoz; Ara arabinoz; Xyl ksiloz Man mannoz, galaktoz Gal GLC glikoz; inositol internal standart.

<ol>
	<li>Albersheim, 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+method+for+the+analysis+of+sugars+in+plant+cell+wall+polysaccharides+by+gas-liquid+chromatography.">A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography.</a> Carbohydr. Res. 5, 340-340 (1967).</li><li>Carroll, A., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+Biofuels.">Cellulosic Biofuels.</a> Annu Rev Plant Biol. 60, 165-165 (2009).</li><li>Foster, C. E., Martin, T., Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+compositional+analysis+of+Plant+Cell+Walls+(Lignocellulosic+biomass),+Part+I:+Lignin.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part I: Lignin.</a> Journal of Visualized Experiments. , (2010).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54 (4), 559-559 (2008).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+approach+to+understanding+plant-cell+walls.">Toward a systems approach to understanding plant-cell walls.</a> Science. 306 (5705), 2206-2206 (2004).</li><li>Teeri, T. T., Brumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterisation+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endotransglycosylases.">Discovery, characterisation and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endotransglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li><li>UPDEGRAF, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEMIMICRO+DETERMINATION+OF+CELLULOSE+IN+BIOLOGICAL+MATERIALS.">SEMIMICRO DETERMINATION OF CELLULOSE IN BIOLOGICAL MATERIALS.</a> Anal. Biochem. 32 (3), 420-420 (1969).</li></ol>

Açıklanan yöntemlerden lignocellulosic bitki biyokütle kompozisyon hızlı bir nicel değerlendirmesini sağlar. Yani matris polisakkaritler hemiselülozlar şeker kompozisyonu, kristalin selüloz içeriği de dahil olmak üzere, bu malzemelerin bileşimi belirleme yöntemi sağlar. Kişi başına düşen çeşitli analitik yöntemlerin hacmi değişir. Burada açıklanan protokolleri kullanarak, 20 örnekleri kristal selüloz içeriği için matris polisakkarit kompozisyonlar ve 30 işlenebilir. Nicel veri en uygun hammadde bitkileri doğası nedeniyle, çeşitli veya genotipler, biyoyakıt üretimi için kendi uygunluk açısından değerlendirilebilir.

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Trifluoroacetic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T6508</td>
<td>&nbsp;</td>
<tr>
<td>myo-Inositol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I5125</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Borohydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>213462</td>
<td>&nbsp;</td>
<tr>
<td>Pyridine</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3348-01</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Anhydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>320102</td>
<td>&nbsp;</td>
<tr>
<td>Spectromax Plus 384</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Plus384</td>
<td>&nbsp;</td>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>7890A GC/5975C MSD</td>
<td>&nbsp;</td>
<tr>
<td>5.5mm Stainless Steel Balls</td>
<td>&nbsp;</td>
<td>Salem Ball Company</td>
<td>(N/A)</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate heat spreader</td>
<td>&nbsp;</td>
<td>Biocision</td>
<td>Coolsink 96F</td>
<td>&nbsp;</td>
<tr>
<td>Retsch Mill</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>TissueLyser II</td>
<td>&nbsp;</td>
<tr>
<td>Heating block</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>Dri-block DB-3D</td>
<td>&nbsp;</td>
<tr>
<td>Sample concentrator</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>FSC400D</td>
<td>&nbsp;</td>		</tbody>
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comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part ii: carbohydrates

Yenilenebilir, karbon nötr, sanayi ve toplum için sürdürülebilir hammadde ihtiyacını 21. yüzyıl için en önemli sorunlardan biri haline gelmiştir. Bu ulaşım için sıvı yakıtların üretimi için endüstriyel hammadde olarak bitkisel ürünlerin kullanımı ilgi başlattı.<sup&gt; 2</sup&gt; Biokompozit malzemeleri gibi ve diğer ürünler<sup&gt; 6</sup&gt;. Bitki biyokütle, gezegen üzerindeki en büyük enerji rezervlerinin biri olmaya devam ediyor<sup&gt; 4</sup&gt;. Bu daha çok enerji selüloz dahil olmak üzere zengin polimerler, çeşitli hemiselüloz ve polifenol lignin oluşan hücre duvarlarının oluşur<sup&gt; 5</sup&gt; Ve bu nedenle bazen lignocellulosics olarak adlandırılır. Ancak, bitki hücre duvarları, duvarları, tesisin tamamının gücü ve yapısal bütünlüğünü yoğun katkıda bozulmaya karşı inatçı evrildi. Için gerekli sertliğin rağmen, hücre duvarı, metabolik olarak aktif ve bitki büyüme ve farklılaşma gibi sayısız hücre faaliyetlerinde hayati roller oynamaktadır son derece dinamik bir varlıktır<sup&gt; 5</sup&gt;. Duvarları çeşitli işlevleri nedeniyle, büyük bir yapısal çeşitliliği, tek bir bitki içinde, farklı bitki türlerinin ve hücre tiplerinin duvarları içinde var<sup&gt; 4</sup&gt;. Bu nedenle, bitki türleri, bitki çeşitliliği, bitki doku biorefinery için ne bağlı olarak, kimyasal / enzimatik süreçleri ve sıvı biyoyakıtlar için çeşitli şekerler sonraki fermantasyon depolymerisation işleme adımları ayarlanabilir ve optimize edilmesi gerekir. Bu gerçek temelini oluşturan bitki biyokütle hammadde tam bir karakterizasyonu için ihtiyaç. Burada lignocellulosics bileşimi tespitine olanak sağlar ve yüksek verimlilik analizi (Şekil 1) bir orta için uygun, kapsamlı bir analitik yöntem açıklanmaktadır. Bu yöntem destarched hücre duvarı materyalinin hazırlanması ile başlar. Ortaya çıkan lignocellulosics sonra hemiselülozlar monosakkarit kompozisyon ve diğer matriks polysaccharides1 ve kristalin selüloz içeriği belirlemek için ayrıldı.<sup&gt; 7</sup&gt;. Lignocellulosic biyokütle lignin bileşenleri analiz etmek için protokol Bölüm tartışıldı<sup&gt; 3</sup&gt;.

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Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Bitki biyokütle, biyoyakıt üretimi için kullanılan olabilir önemli bir karbon-nötr yenilenebilir bir kaynaktır. Bitki biyokütle esas hücre duvarları, lignocellulosics olarak adlandırılan bir kompleks yapılara sahip kompozit malzemeden oluşmaktadır. Burada içerik ve duvar türetilen karbonhidrat bileşimi kapsamlı bir analiz için bir protokol.

Bitki Hücre Duvarları Kapsamlı Kompozisyonel Analiz (Lignocellulosic biyokütle) Bölüm II: Karbonhidratlar

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

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Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

Unit 1

Plant Cell Structure and Organization

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30.0K Görüntüleme.  Michigan State University (MSU). Bitki biyokütle, biyoyakıt üretimi için kullanılan olabilir önemli bir karbon-nötr yenilenebilir bir kaynaktır. Bitki biyokütle esas hücre duvarları, lignocellulosics olarak adlandırılan bir kompleks yapılara sahip kompozit malzemeden oluşmaktadır. Burada içerik ve duvar türetilen karbonhidrat bileşimi kapsamlı bir analiz için bir protokol.

Video: Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Plasenta Hematopoetik Kök Hücre İzolasyonu ve Analizi

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Biyoloji

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Part 2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 3

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

İnsan Pankreas Adacık İzolasyonu: Bölüm II: İnsan Islets saflaştırılması ve Kültür

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Bitki protoplast Floresan Aktif Hücre Ayırma

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

Bitki Hücre Duvarları kapsamlı Kompozisyonel Analizi (Lignocellulosic biyokütle) Bölüm I: Lignin

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Etiket- In situ</emBitki Hücre Duvarlar Lignification> Görüntüleme

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Mikroarray&#39;ler kullanarak Bitki Hücre Duvarı Polimerlerin glukanıdır Profilleme

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Mukayeseli Proteomik Analizi Metabolik Etiketleme ve Membran Fraksiyonasyon<em&gt; Arabidopsis thaliana</em&gt; Süspansiyon Hücre Kültürleri

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...