이 작품은 미국 국립 과학 재단 (National Science Foundation)에서 크리스토프 베닝을 부여하여 지원됩니다.

1. 지질 추출 <ol><li> 한천의 성장 식물에서 4 주 된 Arabidopsis 잎 중간이나 흙을 확정하고 1.5 ML의 폴리 프로필렌 반응 튜브에 그들을 전송 30 MG를 수확하여 지질 추출을 시작합니다. 신선한 잎은는 플래시 액화 질소에 냉동 및 -80 ° C.에 저장할 수 있습니다 </li><li> , 클로로포름과 개미의 산성 (20시 10분 1초, V / V / V) 각 시료에 메탄올로 구성된 용매 300 μL 추출을 추가합니다. 5 분 (페인트 통 또는 이와 유사한 사용) 적극적으로 흔들어. </li><li> 0.2 M의 인산 (H 3 PO 4), 1 M의 칼륨 염화물 (KCl)와 소용돌이 간략 150 μL를 추가합니다. </li><li> 1 분 실온에서 13,000 XG에서 원심 분리기. 낮은 클로로포름 단계에 녹아있는 Lipids는 TLC 플레이트에 발견됩니다. </li></ol> 2. 얇은 레이어 크로마 토그래피 (TLC) 15 <ol><li> 잠수함 0.15 M의 암모늄 황산 ((NH 4) 2 SO 4) 용액에 30 초에 대한 스트립을 읽어와 20cmx20cm 실리카 겔 코팅 TLC 판을 TLC 번호판을 준비하려면 30 초 동안 잠수함이 잠수 후 최소 2 일 동안 번호판을 건조 대상 컨테이너. 인증하는 동안 암모늄의 승화는 다른 glycerolipids로부터 분리에 필요한 phosphatidylglycerol을 protonates 황산, 뒤에 잎. </li><li> 실험 당일, 활성화 120에서 오븐에 굽고하여 번호판을 TLC 2.5 시간에 대한 ° C. </li><li> 상온에 활성 접시를 냉각 후, 크로마토 그램의 기원에 접시에 걸쳐 직선 (접시의 가장자리에서 1.5 cm)을 그리는 연필을 사용합니다. </li><li> 펌 두건에서, 천천히 N 2의 느린 흐름에 따라 200 μL 노란색 플라스틱 팁을 20 μL 피펫을 사용하여 낮은 클로로포름 단계 3 X에게 지질 추출물 20 μL를 제공합니다. 이를 위해 Tygon 튜빙은 N 2 탱크 조절기에 연결되어 있습니다. 지름 1cm보다 작은 자리를 유지. 각 판은 10 샘플 (후속 GLC 분석이 계획되어 경우)까지 저장할 수 있습니다. </li><li> 지방질이 퓸 후드 완전히 건조 명소로서, 아세톤, 톨루엔, 물 (: 30 ML : 7.5 ML 91 ML)로 구성된 개발 용매를 준비합니다. 주위 공기 상대 습도가 높으면 분리에 영향을 수 있습니다. 원하는 분리를 달성하기이 경우 물을 (7.0 ML : : 30 ML 91 ML)를 제공하기 위해 감소되어야합니다. </li><li> 아래로 향하게 샘플 엔드와 탱크에 장소 플레이트 챔버 (H : W = 27.0 : 26.5 7.0 cm / cm / cm L) 개발 sealable TLC에 용매 개발 80 ML 하거라. 클램프를 사용하여 탱크를 밀봉합니다. 솔벤트는 접시를 계승하고 lipids가 분리됩니다. 개발 시간은 실온에서 약 50 분 거리에 있습니다. </li><li> 용매 전면 판의 상단에서 1cm에 도달했습니다 때, 신중하게 탱크에서 접시를 제거하고 약 10 분 퓸 후드 완전히 건조. </li><li> TLC로 구분 Lipids도 reversibly 정량 분석​​ 또는 황산 또는 α - 나프톨와 irreversibly 스테인드에 대한 요오드와 함께 간략하게 물들일 수 있습니다. <ol><li> 황산의 차링 : 15 분 (그림 2A) 120 ° C에서 퓸 후드 구워에서 유리 스프레이 병에 물이 50% 황산과 스프레이 접시. </li><li> glycolipids에 대한 α - 나프톨 얼룩 : 120 ° C에서 2.4 % (W / V) 10 % α - 나프톨 (V / V) 황산, 80 % (V / V) 에탄올 구워 함께 접시에 스프레이 3-5위한 glycolipid 밴드까지 분 (그림 2B) 분홍색이나 보라색 얼룩이 있습니다. Overtreatment은 시약에 황산의 존재로 인해 모든 lipids의 차링으로 이어질 것입니다. </li><li> 요오드 얼룩 : 퓸 후드에 요오드 결정이있는 폐쇄 TLC 탱크 (lipids 볼 때까지 요오드 증기와 분위기의 채도로 이어지는 하단에 트레이)에 접시를 놓습니다. 너무 오래 요오드가 covalently 고도 불포화 지방산 (그림 2C)을 수정할 수 있으므로 요오드에 접시를 노출시키지 마십시오. 또는, 샘플 차선과 interspersed는 표준 차선이 유리 울 N 2는 각각 표준 차선 이상의 폭격을 받았고이를 통해 요오드 결정과 파스퇴르 피펫을 연결을 사용하여 스테인드해야 lipids의 산화를 방지합니다. </li></ol></li></ol> 3. 지방산 아실 Methylester (명성) 반응 16 <ol><li> 면도날과 TLC 판에서 실리카 주변 식별 지질 명소를 제거합니다. 지질이 포함된 실리카을 다쳤고과 테플론 (PTFE) - 줄지어 나사 마개와 유리관에 깔때기를 사용하여 실리카 분말을 전송할 수 있습니다. </li><li> 유리 피펫 각 샘플에 무수 메탄올 1 ML 1 N 염산 (HCL)을 추가합니다. </li><li> 200 μL 노란색 플라스틱 팁과 200 μL 피펫을 사용하여 내부 표준으로 각각의 시료 200 μL 피펫을 사용하여 100 μL 50 μg ML -1 pentadecanoic 산성 (15시)를 추가합니다. 튜브 컨트롤로 methanolic HCL에서만 pentadecenoic 산성과 함께 보관하십시오. 테플론 - 줄지어 모자와 밀접하게 유리 튜브를 닫습니다. </li><li> 부화 GL25 분 80 ° C의 물을 욕조에 엉덩이가 튜브. 용매가 증발하지 않도록 튜브 봉인해야합니다. </li><li> 튜브가 냉각되면 적극적으로 한 ML의 헥산과 와동 다음 1 ML 0.9 %의 나트륨 염화물을 추가합니다. 3 분 1000xg에서 샘플을 원심 분리기. </li><li> 펌 모자, 파스퇴르 피펫으로 시료의 헥산 / 상위 레이어를 제거하고 새로운 13x100 mm의 유리관에 넣습니다. </li><li> 완전히 건조하지 않고 N 2의 느린 흐름에 따라 헥산을 증발. </li><li> 60 μL 헥산의 결과 지방산 아실 methylesters s를 디졸브. autosampler 튜브와 긴밀하게 모자에 샘플을 전송합니다. 샘플 ° C 단기 및 -20 ° C에 대한 몇 일 동안 4 저장할 수 있습니다. </li></ol> 4. 기체 - 액체 크로마 토그래피 (GLC) 10 <ol><li> GLC를 시작하기 전에, 헬륨, 수소와 공기 실린더가 가득 있는지 확인합니다. </li><li> 충분한 헥산은 용매 저수지에 추가되어야하고 폐기물 컨테이너가 비어 있어야합니다. 지방산 아실 methylesters 분리 들어, 시스템에 DB - 23 컬럼을 연결합니다. </li><li> autosampler에 플레이스 유리병. 시스템 컴퓨터에 GLC에 대한 Chemstation 소프트웨어를 시작합니다. </li><li> 250 ° 헬륨 유량 48.6 ML 분 -1에서 속도와 21.93 PSI의 압력 C.에서 입구 온도를 설정 분할 비율은 30.0입니다 : 1. </li><li> 오븐 온도는 2 분 140 ° C에서 초기 설정 ° C 25 ° C 분 -1의 속도로 160 증가합니다. 다음 160의 증가로 온도를 설정할 ° C 250 ° C 8 속도 ° C 분 -1 250에서 개최 ° 140 감소 뒤에 4 분에 대한 C ° C 38 ° C 비율 분 - 1. 하나의 실행 약 21 분 정도 소요됩니다. </li><li> 불꽃 이온화 검출기의 온도는 270입니다 ° 30.0 ML 분 -1, 400 ML 분 -1과 헬륨 유량 30.0 ML 분 -1시에서 공기 흐름 속도의 수소 유량과 C. </li><li> 튜브 및 실행 시퀀스 테이블에 샘플 이름의 번호를 입력합니다. 유리병 당 2 μL 시료를 주입하기 위해 10 μL 주입기를 설정합니다. </li><li> 악기가 준비되면, 실행 순서를 시작합니다. </li></ol> 5. 대표 결과 : 4 주 된 Arabidopsis의 모종의 TLC로 구분 lipids의 돌이킬 수없는 얼룩의 예는 그림 2에 표시됩니다. 황산 스테인드 lipids (그림 2A)가 탄과 갈색 반점으로 나타납니다 있습니다. α - 나프톨 다른 극지 lipids은 (그림 2B) 노란색 얼룩 동안 α - 나프톨은 분홍색 - 보라색 색상을 가지고 물들일 등 Glycolipids을 SQDG, DGDG 같은 MGDG 같은 glycolipids을 얼룩이 좋습니다. 요오드 얼룩도 가능해과 lipids에게 요오드 증발 (그림 2C)로 짧은 시간이 지남에 따라 사라집니다 노란 색상을 제공합니다. 흠없는 lipids가 lipids의 분해 감소 것이 바람직 있지만 간단히 요오드 스테인드 lipids는 GLC 분석의 대상이 될 수 있습니다. 성공적으로 완료하면, 다른 지방산 아실 methylester를 나타내는 독특한 신호는 (그림 3) GLC 후 관찰됩니다. 짧은 탄소 사슬과 적은 더블 채권과 지방산 아실 methylester은 DB - 23 컬럼을 사용하여 짧은 체류 시간이 있습니다. 지방산 아실 methylester 프로 파일링이 변경 지질 성분과 돌연변이를 식별하는 중요한 도구입니다. 그림 4에서 MGDG18 : 3 지방산 어금니 비율은 야생 유형 18 비교 tgd4 - 1 돌연변이 감소됩니다. 모든 지질 클래스의 몰스 하나의 지질 클래스 지방산 아실 methylester의 몰스을 나누어 각 지질의 어금니 비율 계산됩니다. 예를 들어, MGDG의 어금니 비율을 계산 : (MGDG) 몰 %는 = Σ [FAMEs (MGDG)] / Σ [FAMEs (총)] x100의 % 야생 유형과 돌연변이 모두에서 각 지질 클래스의 결과 어금니 비율을 비교할 수 있습니다. 예를 들어, tgd4 - 1 돌연변이 MGDG와 PG의 상대적인 양의 증가하지만 DGDG 및 PE (그림 5) 18 양을 감소했다. <img src="/files/ftp_upload/2518/2518fig1.jpg" alt="그림 1" /> Arabidopsis 모종을 사용하여 극성 지질 분석 그림 1. 플로우 차트. 총 lipids 4 주 된 Arabidopsis의 모종에서 추출한 및 TLC로 구분됩니다. 구분 lipids는 GLC 분석 다음 transesterification에 대한 TLC 플레이트에서 까진 수 있습니다. <img src="/files/ftp_upload/2518/2518fig2.jpg" alt="그림 2" /> 그림 2. TLC 플레이트에 lipids의 분리. 35 MG의 지질 추출물는 (신선한 무게) 야생 유형 모종은 TLC로 구분하여 황산 (A), α - 나프톨 (B) 또는 요오드 증기 (C)로 물들어있다. 세 반복은 각 얼룩 방법으로 표시됩니다. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol, PC, phosphatidylcholine, PE, phosphatidylethanolamine, PG, phosphatidylglycerol, PI, phosphatidylinositol, SQDG, sulfoquinovosyldiacylglycerol. <img src="/files/ftp_upload/2518/2518fig3.jpg" alt="그림 3" /> 그림 3. 지방산 Methylesters의 GLC 분석 (FAMEs)은 야생 유형의 MGDG에서 파생. FAMEs는 30m 모세관 컬럼을 분리하여 불꽃 이온화에 의해 감지됩니다. Pentadecanoic 산성 (15시)는 내부 표준으로 사용됩니다. <img src="/files/ftp_upload/2518/2518fig4.jpg" alt="그림 4" /> 그림 4. 야생 유형 Col2 (흰색 열)과 tgd4 - 1 돌연변이 (검은 열)에 MGDG의 지방산 프로파일. 지방산은 이중 채권의 수에 따라 탄소의 번호로 제공됩니다. 세 반복은 평균이며, 표준 편차가 표시됩니다. <img src="/files/ftp_upload/2518/2518fig5.jpg" alt="그림 5" /> 그림 5. 야생 유형 Col2 (흰색 열)의 폴라 lipids 구성 및 tgd4 - 1 돌연변이 (검은 열). 세 반복은 평균이며 표준 편차는 오류 막대로 표시됩니다.

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J. 50, 679-679 (1952).</li><li>Jordan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+cyanobacterial+photosystem+I+at+2.5+A+resolution.">Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution.</a> Nature. 411, 909-909 (2001).</li><li>Kobayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type-B+monogalactosyldiacylglycerol+synthases+are+involved+in+phosphate+starvation-induced+lipid+remodeling,+and+are+crucial+for+low-phosphate+adaptation.">Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation.</a> Plant J. 57, 322-322 (2009).</li><li>Lu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+ATPase+protein+of+Arabidopsis,+TGD3,+involved+in+chloroplast+lipid+import.">A small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid import.</a> J. Biol. Chem. 282, 35945-35945 (2007).</li><li>Ohlrogge, J., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+biosynthesis.">Lipid biosynthesis.</a> Plant Cell. 7, 957-957 (1995).</li><li>Stahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatography;+methods,+influencing+factors+and+an+example+of+its+use.">Thin-layer chromatography; methods, influencing factors and an example of its use.</a> Pharmazie. 11, 633-633 (1956).</li><li>Stoffel, W., INSULL, W., AHRENS, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-liquid+chromatography+of+highly+unsaturated+fatty+acid+methyl+esters.">Gas-liquid chromatography of highly unsaturated fatty acid methyl esters.</a> Proc. Soc. Exp. Biol. Med. 99, 238-238 (1958).</li><li>Welti, R., Wang, X., Williams, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+tandem+mass+spectrometry+scan+modes+for+plant+chloroplast+lipids.">Electrospray ionization tandem mass spectrometry scan modes for plant chloroplast lipids.</a> Anal. Biochem. 314, 149-149 (2003).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+trafficking+between+the+endoplasmic+reticulum+and+the+plastid+in+Arabidopsis+requires+the+extraplastidic+TGD4+protein.">Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein.</a> Plant Cell. 20, 2190-2190 (2008).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+the+TGD1+chloroplast+envelope+protein+affects+phosphatidate+metabolism+in+Arabidopsis.">Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.</a> Plant Cell. 17, 3094-3094 (2005).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+permease-like+protein+involved+in+ER+to+thylakoid+lipid+transfer+in+Arabidopsis.">A permease-like protein involved in ER to thylakoid lipid transfer in Arabidopsis.</a> EMBO J. 22, 2370-2370 (2003).</li></ol>

관심 없음 충돌 선언하지 않습니다.

GLC와 TLC 결합하여 식물의 극지 lipids의 정량 분석​​을위한 강력하고 신속한 도구를 제공합니다. 지질 구성에 약간의 변화는 확인할 수 있으므로이 방법은 북극 지질 대사 경로 1,20에서 장애인 돌연변이의 대규모 심사에 사용되고 있습니다. 이 방법은 널리 기판으로 극지 lipids을 이용한 효소의 활동을 모니터링하는 데 사용됩니다. 2,6,7 단풍 게다가, 같은 뿌리와 씨앗이나 chloroplasts 및 mitochondria와 같은 subcellular 분수와 같은 다른 식물 조직의 지질 조성도 같은 방법으로 결정하실 수 있습니다. 여기에 사용되는 용매 시스템 (아세톤, 톨루엔, 물) 공장에서 glycolipids 및 phospholipids의 분리에 최적입니다. tetragalactosyldiacylglycerol는 PC에서 실행하는 동안 그러나, tgd1, 2,3,4 돌연변이 및 절연 chloroplasts에서 TGDG은 PE와 함께 실행됩니다. 이 경우에는 클로로포름, 메탄올, 초산과 물 (85 : 20 : 10 : 4, V / V / V / V)와 용매 시스템은 13 사용됩니다. 때로는 두 개의 서로 다른 용매 시스템을 사용하여 2 차원 TLC은 추가 별도의 glycolipids 및 phospholipids 19 수행됩니다. 또한, 식물의 조직 직접 TLC 5 초기 분리하지 않고 전체 지방산 프로파일을 결정하는 GLC 다음 명성 반응을 받다 수 있습니다. 시연 TLC - GLC 시스템 게다가, 지질 프로파일에 사용되는 다른 방법은 직접 전기 분무 이온화 질량 분석계 협동 17 기반으로합니다. 이 방법에서는 추출에 lipids의 초기 색층 분리가 생략됩니다. 그러나,이 방법은 실험실이나 돌연변이 검사에 대한 일상적인 분석을 위해 덜 유용하게 고가의 장비와 숙련된 인력을 필요로합니다.

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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>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)

환경에서 생물 세포막 분리 세포. 이러한 phosphatidylcholine 또는 phosphatidylethanolamine, 셀과 주변 사이의 화학 교류 모두 경계와 인터페이스 역할을 양식 이중층의 점막과 같은 다세포 식물과 동물, glycerolipids에 하나의 세포에서. 동물과 달리 식물 세포는 광합성을위한 특별한 organelle, 엽록체 있습니다. monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) 4 : 엽록체의 복잡한 멤브레인 시스템 즉 고유 glycerolipids 인이 부족해 glycolipids가 포함되어 있습니다. 이러한 lipids의 역할은 단순히 구조적인 이상입니다. 이러한 glycolipids 및 기타 glycerolipids는 photosystem I 및 II는 광합성 8,11에서 glycerolipids의 참여를 나타내는의 결정 구조에서 발견되었습니다. 인산염 기아 동안 DGDG는 phospholipids 9,12의 손실을 보상하기 위해 extraplastidic 세포막로 전송됩니다. 대부분 이러한 lipids의 생합성 및 기능을 우리의 지식은 Arabidopsis thaliana의 14 유전과 생화 학적 연구의 조합에서 파생되었습니다. 이러한 연구하는 동안, 북극 lipids의 분석을위한 간단한 절차는 지질 돌연변이의 검사 및 분석을위한 필수되었으며 자세하게 설명됩니다. 잎 지질 추출물 먼저 얇은 층 크로마 토그래피 (T​​LC)로 구분되며 glycerolipids는 요오드 증기로 reversibly 스테인드 있습니다. 개인 lipids은 TLC 판에서 스크랩한 및 불꽃 이온화 검출 (FID - GLC) (그림 1)과 함께 기체 - 액체 크로마 토그래피로 분석 아르 지방산 아실 methylesters (FAMEs)로 변환됩니다. 이 방법은 돌연변이 검사에 대한 안정적인 도구로 입증되었습니다. 예를 들어, tgd1, 2,3,4 endoplasmic reticulum - 투 - plastid 지질 인신 매매 돌연변이가 비정상 galactoglycerolipid의 축적을 기반으로 발견되었습니다 : trigalactosyldiacylglycerol (TGDG)와 18시 3분의 상대적 금액이 감소 (탄소 더블 채권) 멤브레인 lipids 3,13,18,20의 지방산 아실 그룹. 이 방법은 기판을 6으로 lipids를 사용하여 단백질의 효소 활동을 결정하는 적용됩니다.

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

극지 지질 추출물의 조성과 개별 glycerolipids의 지방산 조성은 간단하고 강력한 지질 프로파일 실험에서 결정됩니다. 이를 위해 glycerolipids는 얇은 층 크로마 토그래피에 의해 격리되고 그들의 아실 그룹의 transmethylation를 받게. 지방산 아실 methylesters는 기체 - 액체 크로마 토그래피에 의해 계량입니다.

씬 크로마 토그래피 (TLC)에 의해 Arabidopsis thaliana 폴라 Glycerolipid의 프로파일은 기체 - 액체 크로마 토그래피 (GLC)와 결합

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

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Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

36.6K 조회수.  Michigan State University. 극지 지질 추출물의 조성과 개별 glycerolipids의 지방산 조성은 간단하고 강력한 지질 프로파일 실험에서 결정됩니다. 이를 위해 glycerolipids는 얇은 층 크로마 토그래피에 의해 격리되고 그들의 아실 그룹의 transmethylation를 받게. 지방산 아실 methylesters는 기체 - 액체 크로마 토그래피에 의해 계량입니다.

비디오: Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

든 샘플 준비 : 울트라 씬 레이어 방법

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

연구

생물학

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

14 일 된 모종의 조직에서 Protoplasts의 분리 Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

루시페라제 Immunoprecipitation 시스템 (입술)에 의해 항체 프로파일

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

불안 정한 다중 단백질 단지의 분리에 의해 생체내에 제어 세포 간 연결 및 단백질 자기 동질 크로마 토그래피

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

에서 외과를 이용한 셀 타입 특정 표현식 프로파일을 사용하여 조직 및 장기 특정 Phytochrome 응답 조사 Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

의 꽃 - 물놀이 변환 Arabidopsis thaliana 검사 pTSO2 : β - glucuronidase 리포터 유전자 발현

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

단백질 정제에서 사용하기 위해 자동화된 소수성 상호 작용 크로마 토그래피 칼럼 선정

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

에서 초기 단계의 배아의 효율적이고 신속한 분리<em&gt; 애기 장대</em&gt; 씨앗

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

의 조직 화학 염색<em&gt; 애기 장대</em&gt; 차 전지 벽 요소

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...