이 작품은 독일 과학 재단 (DFG)와 독일의 연방 및 주 정부의 우수 이니셔티브에 의해 투자되었다.

1. 식물 재료의 Crosslinking <ol><li> 하베스트 1~2g Arabidopsis 50 - ML 플라스틱 튜브에 단풍 (6 10cm의 로제트 직경에) 및 crosslinking 버퍼와 40mL까지 기입니다. </li><li> 단풍이 버퍼 표면 위에 맞춤형 필터 스폰지 권리를 때우는하여 예를 들어, 포장되어있어 있는지 확인합니다. 다음 건조기에 튜브를 삽입합니다. </li><li> 진공은 10 분에 대한 침투. 각 튜브는 crosslinking를 중지 2M 글리신의 2.5mL를 추가하고, 글리신의 동일한 분산을 보증하기 위해 튜브를 반전. </li><li> 진공 다른 5 분에 대한 침투와 체에 잎을 수확하는 동안 액체를 폐기하십시오. </li><li> 워시는 다음 유리 비커에 물 1L, 철저하게 종이 타올로 건조 잎 두 잎. </li><li> 새로운 플라스틱 튜브에 잎사귀를 수집하고 액체 질소에 냉동. 스토어 -80 시에 출발 ° C까지 염색질 절연. </li></ol> 2. 염색질의 분리 <ol><li> 단풍은 사용까지 액체 질소에 보관되었습니다 박격포와 암술과 지상되었습니다. </li><li> 50 - ML 플라스틱 튜브에 분말을 전송하고 30mL 추출 버퍼 # 1 일시 중지합니다. </li><li> 오버헤드 흔드는에 4 ° C에서 15 분에 대해 품어. </li><li> 새로운 50 ML 플라스틱 튜브에 miracloth 네 가지 레이어를 통해 여과물 서스펜션. </li><li> 4 2,800 XG에서 20 분에 대한 회전 ° C. </li><li> 뜨는 제거하고 1mL 추출 버퍼 # 2에서 페인트 브러시와 펠렛을 일시 중지합니다. 처음엔 페인트 브러시와 펠렛을 중지하고 버퍼의 나머지 부분을 추가, 버퍼 # 2의 작은 볼륨을 추가합니다. </li><li> 4 12,000 XG에서 10 분에 1.5mL microfuge 관과 스핀으로 정지를 전송 ° C. </li><li> 한편, 1.5mL 추출 버퍼 # 3를 포함하는 2mL - microfuge 튜브를 준비합니다. </li><li> 스펀 튜브 (2.7 단계)에서 뜨는 제거하고 추출 버퍼 # 3 300μL의 펠렛을 일시 중지합니다. 첫째, 버퍼 # 3의 작은 볼륨을 추가 페인트 브러시와 펠렛을 중지하고 나머지 버퍼를 추가합니다. </li><li> 조심스럽게 pipet 단계 2.8 준비 1.5 ML 추출 버퍼 # 3 위에 펠렛을 (단계 2.9) 중지. 두 단계가 혼합되지 않습니다 있는지 확인합니다. 4 16,000 XG에서 1 시간을 위해 스핀 ° C. </li><li> 뜨는 제거하고 sonication 버퍼의 300μL의 페인트 브러시와 염색질 펠렛을 일시 중지합니다. </li><li> 약 400bp의 DNA를 크기에, sonotrode와 함께, 또는 초음파 욕조에서 염색질을 Sonicate. sonicating 때, 반드시 염색질이 약 30 이상 가열되지 않습니다 ° C는 열에 민감한 crosslinks를 보호하기 위해합니다. sonication의 기간은 다른 sonication 장치 사이에 상당히 차이가 있기 때문에, 실험적으로 결정되어야합니다. 지도 들어, 두 개의 다른 장치에 대한 설정이 제공됩니다 : </li></ol> 0.6mL microfuge 튜브에 Bandelin 브랜드 sonotrode 노출 염색질 25 % 파워 사이클 = 50 설정으로 다섯 번 20 파열과 sonication하십시오. 얼음 욕조에 매 20 번째 파열 후에 이렇게하는 동안, 쿨 샘플. Diagenode Bioruptor 초음파 욕조와 sonication을 위해, 물, 얼음 욕조를 입력하고 &#39;높음&#39;설정을 1.5mL microfuge 튜브에서 30 초 동안 열번 sonicate. 매 30 초 후에 다른 30 ​​초 동안 냉각 샘플. <ol start="13"><li> 스핀은 4 1만6천g에서 5 분에 대한 샘플을 sonicated ° C 소화 되 다만 자료를 촉진합니다. 뜨는 직접 immunoprecipitation 사용할 수 있습니다. 또한, 샘플은 액체 질소에 냉동과 ° C까지 추가로 사용하여 -80에 저장된 충격하실 수 있습니다. </li></ol> 3. Immunoprecipitation <ol><li> 40μL 단백질 - 항체 결합 버퍼의 아가로 오스와 1.8mL.을 포함하는 2 - ML의 microfuge 튜브 준비 염색질의 준비 200μL (2.13 단계)를 추가합니다. </li><li> 오버헤드 흔드는에 1 시간을위한 튜브를 품어. </li><li> 단백질 - 아가로 오스 비즈를 버리고 immunoprecipitation을위한 뜨는 사용합니다. </li><li> 염색질 농도를 ( &#39;입력&#39;)를 확인하는 40μL - 나누어지는을 제거합니다. </li><li> 추가 30μL 단백질 - 아가로 오스와 400μL sonicated 염색질 서스펜션에 특정 시약 및 장비의 아래 테이블에 표시됩니다 수정 - 특정 항체의 금액입니다. </li><li> 4 오버헤드 흔드는에 2.5h 또는 야간에 대해 품어 ° C. </li><li> 440 X G.에서 2min을 위해 구슬을 스핀 </li><li> 각 세척 버퍼 900μL (아래 목록 참조) 비드 펠렛 씻으십시오. 각 버퍼 들어, 4 오버헤드 흔드는 ° C에 10 분에 대한 버퍼에 품어 구슬 다음, 440 XG에서 2min에 대한 스핀 뜨는 삭제하고 다음 버퍼를 추가합니다. <ol><li> 낮은 소금 세척 버퍼 </li><li> 고 염분 세척 버퍼 </li><li> LiCl 세척 버퍼 </li><li> TE 세척 버퍼 </li></ol></li><li> 최종 세척 후 완전히 남아있는 버퍼를 제거합니다. </li></ol> 4. histones과 DNA의 드 - crosslinking, 그리고 시켰던 DNA의 정화 <ol><li> 아가로 오스 펠렛 및 단계 3.4에서 40μl &#39;입력&#39;샘플, 소용돌이 믹서에서 5 분을위한 혼합, 회전 샘플 및 incu에 드 crosslinking 버퍼 100μL 추가° C 야간 65 덜다. </li><li> 상업 DNA 또는 PCR 정화 키트와 함께 DNA 정화. </li><li> 일반적으로 최종 열 eluate의 1시 5분 희석의 5μl가 수행하는 충분 기존의 현장 특정 실시간 정량 RT - PCR (RT - qPCR). </li></ol> 5. 버퍼 표 <table border="1"><tbody><tr><td colspan="2"> Crosslinking 버퍼 </td></tr><tr><td> 나트륨 butyrate </td><td> 10mM </td></tr><tr><td> 자당 </td><td> 400mM </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 10mM </td></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> 5mM </td></tr><tr><td> 포름 알데히드 </td><td> 3퍼센트 (V / V) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 추출 버퍼 # 1 </td></tr><tr><td> 나트륨 butyrate </td><td> 10mM </td></tr><tr><td> 자당 </td><td> 400mM </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 10mM </td></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> 5mM </td></tr><tr><td> 완료 테아제 억제제 </td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 추출 버퍼 # 2 </td></tr><tr><td> 나트륨 butyrate </td><td> 10mM </td></tr><tr><td> 자당 </td><td> 250mM </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 10mM </td></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 10mM </td></tr><tr><td> 트리톤 X - 100 </td><td> 1퍼센트 (W / V) </td></tr><tr><td> 완료 테아제 억제제 </td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 추출 버퍼 # 3 </td></tr><tr><td> 나트륨 butyrate </td><td> 10mM </td></tr><tr><td> 자당 </td><td> 1.7M </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 10mM </td></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 2mM </td></tr><tr><td> 트리톤 X - 100 </td><td> 0.15 % (W / V) </td></tr><tr><td> 완료 테아제 억제제 </td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Sonication 버퍼 </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 25mM </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) - NaOH, pH를 8.0 </td><td> 5mM </td></tr><tr><td> SDS </td><td> 0.5 % (W / V) </td></tr><tr><td> 완료 테아제 억제제 </td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 항체 결합 버퍼 </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 50mM </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) - NaOH, pH를 8.0 </td><td> 1mM </td></tr><tr><td> NaCl </td><td> 150mM </td></tr><tr><td> 트리톤 X - 100 </td><td> 0.1 % (W / V) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 낮은 소금 세척 버퍼 </td></tr><tr><td> NaCl </td><td> 150mM </td></tr><tr><td> SDS </td><td> 0.1 % (W / V) </td></tr><tr><td> 트리톤 X - 100 </td><td> 1퍼센트 (W / V) </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) - NaOH, pH를 8.0 </td><td> 2mM </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 20mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 고 염분 세척 버퍼 </td></tr><tr><td> NaCl </td><td> 500mM </td></tr><tr><td> SDS </td><td> 0.1 % (W / V) </td></tr><tr><td> 트리톤 X - 100 </td><td> 1퍼센트 (W / V) </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) - NaOH, pH를 8.0 </td><td> 2mM </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 20mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> LiCl 세척 버퍼 </td></tr><tr><td> 염화 리튬 </td><td> 250mM </td></tr><tr><td> Nonidet - P40 </td><td> 1퍼센트 (V / V) </td></tr><tr><td> 나트륨 desoxycholate </td><td> 1퍼센트 (W / V) </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) - NaOH, pH를 8.0 </td><td> 1mM </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 20mM </td></tr></tbody></table><table border="1"><tbody><tr><td> TE 세척 버퍼 </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) - NaOH, pH를 8.0 </td><td> 10mM </td></tr><tr><td> 트리스 - HCL, pH를 8.0 </td><td> 1mM </td></tr></tbody></table><table border="1"><tbody><tr><td> Decrosslinking 버퍼 </td></tr><tr><td> 트리스 - HCL, pH를 6.8 </td><td> 62.5mM </td></tr><tr><td> NaCl </td><td> 200mM </td></tr><tr><td> SDS </td><td> 2퍼센트 (W / V) </td></tr><tr><td> DTT </td><td> 10mM </td></tr></tbody></table> 6. 대표 결과 : Arabidopsis thaliana PR2 방어 유전자 표현 β를 인코딩항균 활성 12과 1,3 - 글루 카 네이스는 benzothiadiazole (BTH), 식물 호르몬 살리실산 3 합성 아날로그에 의해 유발되었다. 그림 1 PR2 표현의 활성화를 보여줍니다 히스톤에 리신 잔류물의 아세틸화 증가와 관련된 PR2 모터에 H3 및 H4. nucleosome 밀도는 유전자 활성화 13시 감소 때문에, 히스톤 아세틸화 값 히스톤 3 불변량 지역 항체로 얻은 신호 표준되었습니다. <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="그림 1"/> 그림 1 BTH는 Arabidopsis에 PR2 모터에 히스톤 아세틸화을 유도. 식물은 100μM BTH 또는 wettable 분말 컨트롤과 함께 치료를했다. 72h 후, 단풍이 수집되었고 염색질은 고립되었다. 아세틸화 특정 항체 (같은 그림에 표시된)와 강수량 후 얻은 신호는 히스톤 H3의 불변량 도메인에 항체와 석출하여 얻은 신호 표준되었습니다. 시켰던 염색질은 RT - qPCR에 의해 계량했다. 데이터 BTH 치료의 부재에서 히스톤 수정 수준 표준화하고 있습니다.

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+displacement+in+transcription.">Nucleosome displacement in transcription.</a> Genes Develop. 20, 2009-2017 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

프로토콜의 가장 중요한 단계는 재료의 분쇄와 염색질의 sonication, DNA와 histones, 유형 및 잎 소재의 금액의 crosslinking 수 있습니다. 이러한 문제의 자세한 내용을 보려면 심판을 참조하십시오. 9 10. Sonication 및 crosslinking : 400bp 약 조각 sonication 동안 염색질을 방해하는 것이 중요합니다. 부족한 sonication 오래 염색질의 조각을 떠날 것입니다 반면 철저한 sonication은 DNA와 histones을 방해하여 염색질을 파괴합니다. 테스트 현장에서 말초 히스톤 수정을 로컬 변경에서 차별 수 없기 때문에 이들은 방법의 특이성에 영향을줍니다. Sonication 효율이 가장 DNA를 분리하고 아가로 오스 겔 전기 영동에 의해 가지각색으로 DNA의 길이를 결정, 전에 sonication, DE - crosslinking의 DNA와 histones 후 샘플에서 20μL 염색질을 수집하여 테스트됩니다. 염색질 준비가 아직 조각난 DNA의 시각화를 방해할 수도 정화의이 단계에서 RNA의 대량 포함되어 있기 때문에 RNAse는 전기하기 전에 샘플에 추가되어 있어야합니다. 비 가지각색 염색질의 DNA가 10kbp 이상인 경우 가지각색 염색질의 DNA가 DNA의 400bp 주변의 최대 신호 강도와 얼룩을 형성 반면 샘플은 염색질 immunoprecipitation하는 데 유용합니다. 우리는 일상적으로 그대로 단풍의 염색질 crosslinking 3 % (V / V) 포름 알데히드를 사용하지만 1% (V / V) 포름 알데히드는 대부분의 경우 충분 수도 있습니다. 우리 손에, 낮은 포름 알데히드 농도는 결과의 PCR 반응에서 짧은 sonication 시간 및 배경 신호 수 있습니다. 그러나 포름 알데히드의 부족 금액은 자주 독립 실험 사이 PCR 신호의 낮은 재현성의 결과. 이것은 낮은 농도에서 포름 알데히드와 나뭇잎의 부족 침투에 의한 것입니다. 화합물이 시간과 중합하는 경향으로 자주 포름 알데히드 주식을 교환해야합니다. 포름 알데히드 솔루션은 약 한 달 동안 안정하고 있으며,이 기간은 거의 메탄올 안정화에 의해 연장되지 않습니다. 이것은 실험 조건을 설정할 때 다른 포름 알데히드 농도를 테스트하는 것이 좋습니다. 식물 재료의 금액 및 연삭 : 서로를 지키는 나뭇잎을 피하기 위해 버퍼의 큰 볼륨의 잎 소재의 낮은 금액을 침투하는 것이 중요합니다. 나뭇잎이 포름 알데히드의 부족 침투에 자주 결과를 집어넣는. 또한, 너무 많이 식물 재료 miracloth 조직의 모공을 차단합니다. 분쇄 효율이 상당히 완벽하게 맞지과 박격포와 유봉을 사용하여에 따라 달라집니다. 우리는 일상적으로 추가 액체 질소의 부재에 액체 질소 냉각 유봉과 박격포와 함께 갈아 50초 세 번 자료가 벌금 파우더로 그라운드 때까지.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 단백질 - A - 아가로 오스 </td><td> 호슈 </td><td> 11 134 515 001 </td><td> 항체 클래스에 따라 달라집니다 </td></tr><tr><td> 단백질 G - 아가로 오스 </td><td> 호슈 </td><td> 11 243 233 001 </td><td> 항체 클래스에 따라 달라집니다 </td></tr><tr><td> 안티 hyperacetyl - H4 </td><td> Millipore </td><td> 06-946 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 - 아세틸 - H4K5 </td><td> Millipore </td><td> 07-327 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 - 아세틸 - H4K8 </td><td> Millipore </td><td> 07-328 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 - 아세틸 - H4K12 </td><td> Millipore </td><td> 07-595 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 - 아세틸 - H4K16 </td><td> Millipore </td><td> 07-329 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 - 아세틸 - H4K18 </td><td> Millipore </td><td> 07-354 </td><td> 우리는 1μL을 사용 </td></tr><tr><td> 안티 - 아세틸 - H3K9 </td><td> Millipore </td><td> 07-352 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 - 아세틸 - H3K14 </td><td> Millipore </td><td> 07-353 </td><td> 우리는 1μL을 사용 </td></tr><tr><td> 안티 trimethyl - H3K4 </td><td> Diagenode </td><td> PAB - 003-50 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 - 디메틸 - H3K4 </td><td> Millipore </td><td> 07-030 </td><td> 우리는 5μL을 사용 </td></tr><tr><td> 안티 모노 메틸 - H3K4 </td><td> Abcam </td><td> ab8895 </td><td> 2,5 - 10μL </td></tr><tr><td> 안티 H3 </td><td> Abcam </td><td> ab1791 </td><td> 우리는 히스톤 밀도를 확인 1μL를 사용 </td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD - 200 </td><td></td></tr><tr><td> Sonotrode MS72 </td><td> Bandelin </td><td></td><td></td></tr><tr><td> Miracloth </td><td> Calbiochem </td><td> 475855 </td><td></td></tr><tr><td> 완료 테아제 억제제 </td><td> 호슈 </td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

염색질 구조 eukaryotes에서 유전자 발현의 조절에 중요​​합니다. 이 과정에서, histones H3 및 H4의 아미노 터미널 꼬리에 염색질의 리모델링, DNA의 methylation과 공유 결합 수정 필수적 역할 1-2 재생합니다. H3 및 H4 히스톤 수정 리신과 아르기닌의 methylation, 라이신의 아세틸화, 그리고 세린의 잔류물 1-2의 인산화을 포함합니다. 이러한 수정은 유전자 활성화, 탄압, 또는 적절한 신호 (미생물 - 관련 분자 패턴, 빛, 호르몬 등) 3-7의 인식 후 표현보다 신속하고 강력한 인증을 지원하는 유전자의 상태와 준비하는 중 연결됩니다. 여기, 우리는 선택된 식물 유전자에서 특정 염색질 변경의 신뢰성과 민감한 검출을위한 방법을 제시한다. 기술은 수정 - 특정 항체 9,10와 포름 알데히드 8,9, 추출 및 염색질, 염색질 immunoprecipitation (칩)의 sonication과 (수정) histones과 DNA의 crosslinking 기반으로, 히스톤 - DNA의 단지의 드 - crosslinking, 그리고 유전자 특정 실시간 정량 PCR. 접근 방식은 C 네 옥수수 5,11의 광합성과 Arabidopsis 3 체계 면역과 관련된 특정 히스톤 변경을 감지하는 데 유용합니다 입증되었습니다.

Watch this Scientific Journal Video about Detection of Histone Modifications in Plant Leaves at JoVE.com

Detection of Histone Modifications in Plant Leaves

특정 식물 유전자 히스톤 수정을 감지하는 신뢰할 수있는 유용한 접근 방법이 설명되어 있습니다. 접근 방식은 염색질 immunoprecipitation (칩)와 실시간으로 양적 PCR을 결합하여. 그것은 다양한 생리 과정에서 역할과 특정 유전자 히스톤 수정을 감지 수 있습니다.

식물 잎에서 히스톤 수정을 감지

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

detection-of-histone-modifications-in-plant-leaves

Nanyang Technological University

Research

JoVE Journal

Biology

24.8K 조회수.  RWTH Aachen University. 특정 식물 유전자 히스톤 수정을 감지하는 신뢰할 수있는 유용한 접근 방법이 설명되어 있습니다. 접근 방식은 염색질 immunoprecipitation (칩)와 실시간으로 양적 PCR을 결합하여. 그것은 다양한 생리 과정에서 역할과 특정 유전자 히스톤 수정을 감지 수 있습니다.

비디오: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

엘리사에 의해 네이티브 손상 Nucleosomes에 포스트 translational 수정의 감지

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

연구

생물학

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

게이트웨이 가능 Bimolecular의 형광 Complementation (BiFC) 시스템을 사용하여 공장에서 단백질 상호 작용의 검출

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

포유류의 링커 - 히스톤 Subtypes의 표현 분석

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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 ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

식물 단백질 복합체의 번역 후 변형의 확인

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

공장에서 유체 Apoplastic의 추출을위한 침투-원심 분리 기술을 사용하여 잎<em&gt; 위 Phaseolus 심상</em&gt; 예를 들어

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

토마토 털이 뿌리의 생성에 의한 높은 처리량 CRISPR 벡터 건설 및 DNA 수정의 특성

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

히스톤 추출에서 데이터 분석에 : 상향식 질량 분석 사용 히스톤 번역 후 변형 분석을위한 완벽한 워크 플로우

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

하이브리드 컷 : 난 분해성 식물 조직 샘플에 대한 개선 단면 방법

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...