우리는 최고의 기술 서비스와 존 랄프 귀중한 조언, 토론 및 포플러 나무 샘플에 대해 위스콘신 대학의 매튜 로버트 웨더에게 감사하고 있습니다. 이 작품은 과학의 오피스 에너지의 미국학과 (DOE) 대호 Bioenergy 연구 센터 (과학 DE - FC02 - 07ER64494의 DOE BER 사무소)와 화학 과학 Geosciences 및 Biosciences 부문, 기초 에너지 과학 사무소에 의해 재정 지원되었다 에너지 미국학과 (보너스 번호. DE - FG02 - 91ER20021).

1. 세포 벽 절연 <ol><li> iWall를 사용하여 2 ML Sarstedt 스크류 캡 튜브에 5.5 mm 스테인레스 스틸 공과 함께 약 60 70mg 공기 또는 동결 건조 식물 소재를 갈아, 분쇄 및 분배 로봇 (30 S). 볼 - 밀 (retschmill)를 사용하여 대안이 아닌 로봇 낮은 처리량 절차는 2 부 2 제공됩니다. </li><li> 적절 지상 소재, 그리고 철저하게 소용돌이 70 % 에탄올 수용액의 1.5 ML를 추가합니다. </li><li> 펠렛 10 분 알코올 불용성 잔류물을 위해 10,000 RPM에서 원심 분리기. </li><li> 뜨는을 대기음 또는 가만히 따르다. </li><li> 잔류물에 클로로포름 / 메탄올 (1:1 V / V) 솔루션의 1.5 ML를 추가하고 펠렛을 resuspend하기 위해 철저하게 튜브를 흔들. </li><li> 10,000 10 분에 대한 RPM 및 표면에 뜨는를 대기음 또는 가만히 따르다에서 원심 분리기. </li><li> 아세톤 500 μl에 Resuspend 펠릿. </li><li> 건조까지 35 공기의 흐름 ° C로 용매를 증발. 필요한 건조 표본은 추가 처리까지 방 온도에서 저장될 수있다면. </li><li> 샘플에서 전분의 제거를 시작하려면 0.1 M 아세트산 나트륨 버퍼 산도 5.0 1.5 ML의 펠렛을 resuspend. </li><li> 20 분 대한 Sarstedt 튜브와 열을 모자. 80 ° C 가열 블록에. </li><li> 얼음에 정지를 쿨. </li><li> 펠렛 다음 에이전트를 추가합니다 : 0.01 % 나트륨 azide (NaN3) 35 μl, 35 μl 아밀라아제 (50 μg / ML H 2 O, 바실러스 종, 시그마), 17 μl pullulanase (바실러스 acidopullulyticus에서 17.8 단위, 시그마) . 튜브와 철저하게 소용돌이를 캡. </li><li> 정지 37 박 이상 incubated입니다 ° 통에 C. 튜브를 Orienting 수평 보좌관은 혼합 향상. </li><li> 100 열 정지 ° C 소화를 종료하기 위해 가열 블록에서 10 분. </li><li> 원심 분리기 (10,000 RPM, 10 분)와 뜨는 포함 solubilized 전분을 삭제. </li><li> , 1.5 ML 물을 추가 vortexing, centrifuging 및 세척 물을 decanting하여 나머지 펠렛을 세 번 씻으십시오. </li><li> 아세톤 500 μl에 Resuspend 펠릿. </li><li> 건조까지 35 공기의 흐름 ° C로 용매를 증발. 그것은 더 건조 주걱으로 튜브에 재료를 헤어지게하는 것도 필요할 수 있습니다. 마른 재료는 절연 세포 벽 (lignocellulosics)를 제공합니다. 필요한 건조 표본은 추가 처리까지 방 온도에서 저장될 수있다면. </li></ol> 2. 리그닌 함량 이 방법은 후쿠시마와 하트 3보고 방식을 기반으로합니다. <ol><li> 1 무게 - 준비 세포 벽 재료의 1.5 MG (1 참조) 빈에 대한 빈 튜브를 떠나는 두 ML의 판매량 플라스크에 있습니다. </li><li> 튜브의 바닥에있는 세포 벽 자료를 수집하는 아세톤 250 μl와 튜브 벽을 씻어하고, 공기에서 아세톤은 매우 부드러운 증발. </li><li> 부드럽게 물 튀기지을 방지하기 위해 튜브 벽을 따라 신선한 만든 아세틸 브로마이드 솔루션 100 μl (25 % 빙초산에 V / V 아세틸 브로마이드)를 추가합니다. </li><li> 50 캡 판매량의 술병과 열 ° 2 시간을위한 C </li><li> 매 15 분 vortexing과 추가 시간 동안 가열. </li><li> 상온에 얼음 쿨. </li><li> 2M 수산화 나트륨과 신선한 0.5 M 히드록 실 아민 염산염의 70 μl 400 μl를 추가합니다. 와동 용적의 flasks. </li><li> 빙초산, 모자와 정확히 2.0 ML 표시 용적 플라스크를 입력하고 혼합을 여러 번 반전. </li><li> 피펫 200 UV 특정 96 잘 판에 솔루션의 μl와 280nm에서 엘리사 Reader에서 읽어보세요. </li><li> (; 풀밭 = 17.75; 포플러 = 18.21 Arabidopsis = 15.69) 적절한 계수를 사용하는 아세틸 브로마이드 가용성 리그닌 (% ABSL)의 비율을 결정하는 다음 수식과 : % ABSL CALC : <img src="/files/ftp_upload/1745/1745eq.jpg" alt="그림 1" /> UG / MG 세포 벽 단위에서 10 %의 결과와 함께 ABSL의 증식 그것은 적어도 3 판은 입자가 흡광도 값에서 약간의 변화를 일으킬 수 있기 때문에 흡광도 (ABS)을 평균 읽습 할 수 있도록 도와줍니다. 참고 : 0.539 cm가 pathlength를 나타내고 있지만, 플레이트에 따라이 결정해야 할 수도 있습니다. </li></ol> 3. 리그닌 성분 이 방법은 로빈슨과 맨스필드 5 발표된 최근 메서드에서 채택됩니다. <ol><li> thioacidolysis위한 나사 덮인 유리관에 세포 벽 재료의 약 2 MG (1을 참조하십시오.) 전송합니다. </li><li> 조심스럽게 2.5 % 붕소 trifluoride 디에틸 etherate (BF 3) 10 % 에탄티올 (EtSH) 솔루션을 준비합니다. 당신은 질소와 dioxane 병에 잃어버린 볼륨을 치환하기 위해 질소 가스로 가득 풍선을 사용해야합니다. Dioxane 매우 위험한이며, 후드 밖으로 샘플이나 장비를하지 않습니다. 20 μl EtSH,, 5 μl BF 3 175 μl dioxane : 볼륨 샘플 당 솔루션의 준비했습니다. </li><li> EtSH, BF 3, 각 샘플에 dioxane 솔루션을 200 μl를 추가합니다. </li><li> 바로 질소 가스와 모자와 퍼지 유리 상부. </li><li> 100 히트 ° 부드러운 혼합 매 시간과 4 시간 동안은 C. </li><li> 5 분 얼음에 냉각하여 최종 반응. </li><li> 0.4M 나트륨 중탄산염 150 μl, 소용돌이 추가 </li><li> 청소 물 1 ML과 에틸 아세테이트, 소용돌이 0.5 ML를 추가하고 (아래에서 위로, 물, 에틸 아세테이트에) 단계 분리하게하십시오. </li><li> 2 ML Sarstedt 튜브에 에틸 아세테이트 층 150 μl을 전송합니다. 물이 전송이되지 있는지 확인하십시오. </li><li> 공기와 농축기로 용매를 증발. </li><li> (두 번 총 반복 초과하는 물을 제거) 200 μl 아세톤을 추가하고 증발. </li><li> TMS의 derivati​​zation에 대한 에틸 아세테이트 500 μl, 피리딘 20 μl, 그리고 N 100 μl, O - BIS (trimethylsilyl) 각 튜브에 acetamide를 추가합니다. </li><li> 25 2 시간 동안 품어 ° C. </li><li> GC / MS 유리병에 반응 100 μl를 전송하고 아세톤 100 μl를 추가합니다. </li><li> quadrupole 대량 분광계 또는 불꽃 이온화 검출기가 장착된 GC로 샘플을 분석할 수 있습니다. 애질런트 HP - 5MS 컬럼은 (X 0.25 mm X 0.25 μm의 필름 두께 30mm)으로 설치됩니다. 다음 온도 기울기는 30 분 용매 지연과 1.1 ML / 분 유량과 함께 사용됩니다 : 130에서 초기 개최 · 3 분 C, 3 ° 250 C / 분 램프 ° C 1 분 만요, 수 130 ° C.의 초기 온도 평형 </li><li> 봉우리는 tetracosane 내부 표준 (옵션)를 사용하여 상대 유지 시간 또는 299의 특성 질량 스펙트럼 이온에 의해 식별 M / Z, 269m / Z, 그리고 239m / S, G에 대한 Z와 H 단량체, 각각 (그림을 참조하십시오. 2). 리그닌 구성 요소의 구성은 100 %로 총 피크 영역을 설정하여 계량입니다 </li></ol> 4. 대표 결과 벽 분석의 예제는 그림 2에 표시됩니다. 이 경우에는 포플러 줄기 (나무)은 프로토콜 섹션에서 설명한 여러 가지 절차에 의해 분석되었다. thioacidolysis 및 TMS - derivati​​zation 후 리그닌 - 구성 요소의 분리 예제 크로마토 그램이 표시됩니다. 분명의 상대적 풍부한 syringyl - (S), guaiacyl - (G) 및 P - hydroxyphenol - (H) 단위가 결정하실 수 있습니다. 아세틸 브로마이드 가용성 리그닌의 내용은 자체 설명이고, 하나는 벽 건조 무게의 20-50% 사이의 가치를 기대할 수 있습니다. 하나는 아세틸 브로마이드는 벽에 리그닌 현재의 모든 solubilize하고, solubilization의 정도는 물질에 따라 달라질 수 있습니다하지 않습니다한다. 그러나,이 방법은 비교적 수행하는 간단하고 신속하고 리그노셀룰로오스성 물질의 리그닌 내용의 훌륭한 근사치를 제공합니다. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="그림 1" /> 그림 1 :. 리그노셀룰로오스성 분석의 개요 세포 벽 (lignocellulosics)는 원유 건조 식물으로부터 격리됩니다. 벽 재료는 다음 aliquots에 가중치 및 다양한 assays에 대한 세분화됩니다. 월 자료는 아세틸 브로마이드와 UV - 분광하여 계량 solubilized 리그닌과 함께 처리됩니다. 리그닌 성분의 결정에 대한, 벽면 소재는 thioacidolysis를 받게됩니다. solubilized phenolics는 TMS의 derivati​​zation를 받아야하고 분리 및 GC - MS 분석에 의해 계량하실 수 있습니다. 매트릭스 다당류 성분과 결정 셀룰로스 컨텐츠 프로토콜 2 부 2 설명되어 있습니다. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="그림 2" /> 그림 2 :. 포플러 (포풀러스 tremoloides)에서 포플러 나무 종합 리그노셀룰로오스성 분석 우드 칩은 설명 프로토콜을 받게되었습니다. Ligin 조성, H P - 히드록시 페닐, G guaiacyl, S syringyl 단위.

<ol>
	<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> Annual Review of Plant Biology. 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+II:+Carbohydrates.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part II: Carbohydrates.</a> J Vis Exp. , (2010).</li><li>Fukushima, R. S., Hatfield, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+isolation+of+lignin+for+utilization+as+a+standard+to+determine+lignin+concentration+using+the+acetyl+bromide+spectrophotometric+method.">Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method.</a> J. Agric. Food Chem. 49 (7), 3133-3133 (2001).</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>Robinson, A. R., Mansfield, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+poplar+lignin+monomer+composition+by+a+streamlined+thioacidolysis+procedure+and+near-infrared+reflectance-based+prediction+modeling.">Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling.</a> Plant J. 58 (4), 706-706 (2009).</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,+characterization+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endo-transglycosylases.">Discovery, characterization and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endo-transglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li></ol>

설명 방법은 리그닌 함량과 리그노셀룰로오스성 식물 바이오 매스의 구성의 급속한 양적 평가를 가능하게합니다. iWall 로봇을 사용하여 약 350 샘플은 지상과 하루에 적절하게 사용하실 수 있습니다. 인당 다양한 분석 방법의 처리량이 다릅니다. 사용 프로토콜은 여기에 설명된 30 샘플은 리그닌 함량에 대한 처리하고, 하루 리그닌 성분 15 수 있습니다. 데이터 최적의 공급 원료 작물의 양적 특성으로 인해, 다양한이나 genotypes는 생물 연료 생산을위한 그들의 적합성의 관점에서 평가하실 수 있습니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Hydroxylamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>255580</td>
<td>&nbsp;</td>
<tr>
<td>Acetyl Bromide</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>135968</td>
<td>&nbsp;</td>
<tr>
<td>Ethanethiol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E3708</td>
<td>&nbsp;</td>
<tr>
<td>Borontrifluoride diethyl etherate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15719</td>
<td>&nbsp;</td>
<tr>
<td>N,O,-Bis(trimethylsilyl) acetimide</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15241</td>
<td>&nbsp;</td>
<tr>
<td>Dioxane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>296309</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>6890 GC/5975B MSD</td>
<td>(lignin composition)</td>
</tr>
<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>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>
		</table>
		</div>

comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part i: lignin

재생, 탄소 중립, 그리고 산업과 사회에 대한 지속적인 원료의 필요성은 21 세기의 가장 눌러 문제 중 하나가되고있다. 이것은 교통 액체 연료의 생산을위한 산업 원료 등 공장 제품의 사용에 흥미를 rekindled있다<sup&gt; 1</sup이러한 biocomposite 자료 등&gt; 및 기타 제품<sup&gt; 7</sup&gt;. 식물 바이오 매스는 지구상에서 가장 큰 미개척 보유 중 하나 남아있다<sup&gt; 4</sup&gt;. 그것은 주로 에너지 셀룰로오스를 포함한 다양한 고분자 다양한 hemicelluloses (매트릭스 다당류와 리그닌의 폴리페놀 구성되어 있으며, 세포 벽 구성됩니다<sup&gt; 6</sup&gt; 때문에 때로는 lignocellulosics를 칭했다. 그러나, 식물의 세포 벽을 벽에 세포 전체 식물 인장 강도를 제공과 같은 병원체를 구하고, 물이 식물을 통해 이송 수 있도록, 저하에 완강히 저항하는 것으로 진화했습니다;까지 나무의 경우에는 위의 더 100m에 지상 수준. 벽의 다양한 기능으로 인해 엄청난 구조적 다양성은 단일 플랜트 내의 다른 식물 종의 및 세포 유형의 벽 안에있다<sup&gt; 4</sup&gt;. 따라서 농작물 종, 작물 다양성, 또는 식물의 조직이 biorefinery 사용되는 무엇에 따라, 화학 / 효소 프로세스와 액체 바이오 연료에 대한 다양한 설탕의 후속 발효에 의해 depolymerization에 대한 처리 단계 조정하고 최적화해야합니다. 이 사실 뒷받침 식물 바이오 매스 feedstocks의 철저한 특성화를위한 필요합니다. 여기 lignocellulosics의 구성의 결정을 가능하게 높은 처리량 분석 매체 의무가있는 포괄적인 분석 방법을 설명합니다. 첫번째 부분에서 우리는 폴리페놀 리그닌 (그림 1)의 분석에 중점을두고 있습니다. 방법은 destarched 세포 벽 재료 준비와 함께 시작합니다. 그 결과 lignocellulosics은 다음 acetylbromide의 solubilization하여 리그닌 함량을 결정하는 갈​​라 아르<sup&gt; 3</sup그 syringyl, guaiacyl 및 P - 히드록시 페닐 단위의 관점에서&gt;와 리그닌 성분<sup&gt; 5</sup&gt;. 셀룰로스 내용 및 매트릭스 다당류 성분을 포함 리그노셀룰로오스성 바이오 매스의 탄수화물을 분석하는 프로토콜은 부 II에서 설명합니다<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin at JoVE.com

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

식물 바이오 매스는 바이오 연료 생산에 사용될 수있는 주요 탄소 중립 되살릴 수있는 자원입니다. 식물 바이오 매스는 주로 세포 벽, lignocellulosics를 칭했다 구조적으로 복잡한 복합 재료로 구성되어 있습니다. 여기서 우리는 내용과 polyphenolic의 리그닌의 구성의 포괄적인 분석을 위해 프로토콜을 설명합니다.

식물 세포 벽의 종합 작곡 분석 (리그노셀룰로오스성 바이오 매스) 부 I : 리그닌

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 I: Lignin

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.0K 조회수.  Michigan State University (MSU). 식물 바이오 매스는 바이오 연료 생산에 사용될 수있는 주요 탄소 중립 되살릴 수있는 자원입니다. 식물 바이오 매스는 주로 세포 벽, lignocellulosics를 칭했다 구조적으로 복잡한 복합 재료로 구성되어 있습니다. 여기서 우리는 내용과 polyphenolic의 리그닌의 구성의 포괄적인 분석을 위해 프로토콜을 설명합니다.

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

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

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 characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have 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. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

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 I: digestion and collection of pancreatic tissue) using a modified automated method.

인간 췌장 아일릿 격리 : 부품 I : 소화 및 췌장 조직의 수집

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

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생물학

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.

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

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 1 부

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.

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

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 2 부

Vaccinia Virus Infection &amp; 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.

우두 바이러스 감염 및 바이러스 유전자 발현의 시간적 분석 : 제 3

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

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 transportation2 and other products such as biocomposite materials6. 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, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. 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 depolymerisation 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 (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

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

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 wall derived carbohydrates.

식물 세포 벽의 종합 작곡 분석 (리그노셀룰로오스성 바이오 매스) 2 부 : 탄수화물

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.

라벨 - 무료 현장에서</em식물 세포 벽 Lignification의> 이미징

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.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

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.

비교 단백체 분석을위한 신진 대사 라벨링 및 막 분별<em&gt; 애기 장대</em&gt; 서스펜션 세포 배양

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

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

전체 마운트 난자에있는 염색질 수정 및 핵 구조의 정량, 단일 세포 분석을위한 효율적인 방법<em&gt; 애기 장대</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

식물 표피 지질 폴리 에스테르 단량체의 분리 및 성분 분석

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...