NU - 엘리사 방법의 개발은 NIH 부여 RO1AG023687에 의해 지원되었다.

1. 포유 동물 세포 배양 NU - 엘리사는 문화의 성장 수있는 모든 포유 동물 세포 종류에 수행할 수 있습니다. 우리는 따라서 여러 가지 항체 (ABS)와 assays를 허용, nucleosomes가 몇 가지 동일하게 로드된 엘리사 접시를 준비하는 충분한 수량에 격리 수 있도록 세포의 대규모 배치에 중간 준비하는 것을 선호합니다. 다음 문화 비늘은 충분한 자료를 제공합니다 마우스 embyronic의 줄기 세포에 대한, 피더 세포없이 세포 중 하나 또는 두 개의 15cm 번호판을 성장. 섬유아 세포의 경우, 합류로 5-10 15cm 번호판을 성장. 2. 핵의 분리 참고 :로 표시를 제외한 모든 단계, 사전 차게 버퍼와 얼음에 있습니다. <ol><li> 접시 당 3 ML의 트립신과 세포를 Trypsinize, 결합, 그리고 얼음처럼 차가운 PBS / butyrate 20 ML를 추가합니다. 5 분 1,000 rpm으로 원심 분리기. </li><li> 10 ML PBS / butyrate의 세포를 Resuspend 5 분 1,000 rpm으로 원심 분리기. </li><li> 단백 분해 효소 억제제 (시그마 - 알드리치 P - 8340) 4 개 ML의 용해 버퍼에 Resuspend. 얼음 타입 B의 유봉 20 스트로크를 균질 다운스. </li><li> 4 10 분 2천g에서 원심 분리기 ° C. (뜨는이 프로토콜에 대한 필요하지 세포질을 포함하지만, 원하는 경우는 다른 용도로 저장할 수 있습니다.) </li><li> 2 ML 얼음 차가운 씻어 버퍼 C에서 펠렛을 (프로 테아제 억제제와 함께) Resuspend. </li><li> 레이어 5 ML 30 % 자당 쿠션 통해 resuspended 자료는 다음, 스윙 버킷 로터 5 분 2,400그램에서 원심 분리기. 핵은 쿠션을 통해 마이 그 레이션되며, 파편이 인터페이스에 남아 있습니다. </li><li> 신중하게 모든 액체 볼륨을 제거하고 250 μl 얼음 차가운 세척 버퍼 C + 프로 테아제 억제제의 핵 resuspend. </li></ol> 3. 현장 Micrococcal Nuclease (MNase) 소화에 의해 Nucleosomes의 분리 우리는 염색질이 뜨는로 무료 그대로 nucleosomes 운전 hypotonic 치료 다음, MNase 그들을 일으키는 핵 이내에 현장에서 소화되는 프로 시저를 사용하십시오. <ol><li> 샘플 3 μl 0.1 M CaCl 2를 추가합니다. 37 ° C 열 블록에 넣어. 37 ° C 온도를 가정하도록 허용합니다. </li><li> 2 대에게 MNase을 (MNase 버퍼 2 units/10μl에 용해 Micrococcal Nuclease, 시그마 - 알드리치) 추가 후, 37에 대한 품어 ° pipet 팁을 사용하여 자주 혼합 12 분, 대한​​ C. </li><li> 반응을 중지 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) 나 -, 산도 8.0 6 μl를 추가합니다. 얼음 입어. </li><li> 뜨는 폐기, 4 분 2천그램에서 원심 분리기. 300 μl 0.2 MM 나 - EDTA (에틸렌 다이아 민 테트라 초산)에서 펠렛을 Resuspend. 가끔 부드러운 pipeting 1 H 위해 얼음에 보관하십시오. (이 hypotonic 조건이 핵에서 무료 nucleosomes을 해방). </li><li> 4 4 분에 대한 3,000그램에서 원심 분리기 ° C. 얼음, 무료 nucleosomes를 포함 뜨는을 저장합니다. </li><li> 300 μl 0.2 MM 나 - EDTA (에틸렌 다이아 민 테트라 초산)과 함께 다시 펠렛을 Resuspend. 가끔 부드러운 pipeting 1 H 위해 얼음에 보관하십시오. </li><li> 4 4min에 대한 3천그램에서 원심 분리기 ° C. 뜨는을 유지하고 5 단계에서 첫 번째 뜨는와 결합. 그 결과 mononucleosome 준비는 -80 ° C.에 aliquoted하고 저장할 수 있습니다 </li></ol> 참고 : 염색질의 금액 crudely 10 μl 샘플 260 nm의 흡광도에서 측정하여 quantitated 수있는 것은 물이 990 μl에 추가됩니다. 260 = 10 (1백분의 1 희석에 대한 조정 후) 염색질의 약 1mg/ml에 해당합니다. 주로 길이 146 BP의 DNA를 포함해야 MNase digestions의 품질과 범위를 모니터링하기 위해 DNA 내용에 대한 또한, 위의 절차를 수행하는 동안, 작은 샘플이 유지 수 있으며 나중에 분석했다. Laddering 불완전 MNase의 소화 나타내는 것입니다. 참고 : Fig.1은 핵 분리 및 MNase의 digestions 단계의 diagrammed 요약이 포함되어 있습니다. 4. Nucleosome - 엘리사 (NU - 엘리사) 우리는 96 잘 엘리사 microtiter 접시에 고정되었습니다 원시 그대로 nuclesosomes를 포함한 분수에 PTMS를 감지. 각 샘플의 경우, 우리는 2 배 dilutions 일련의를하고, 이러한 세중의에 우물에 코팅됩니다. <ol><li> 4 명이 50 μl / 음의 코팅 버퍼에 희석 nucleosomes와 C.에서 하룻밤 코트 MaxiSorp 플레이트 nucleosomes의 제안 시리얼 이중의 dilutions는 바닥에서만 코팅 버퍼 (0 μg의 염색질)와 0.1 μg, 0.05 μg, 0.025 μg, 0.0125 μg, 0.00625 μg, 0.00313 μg, 0.00156 μg을 추가하여 아래 행 맨 앞줄에서 준비가되어 행. (참고 : 판 표지이 단계가 필요합니다.) </li><li> 아침에 접시 세척기 10 분 결합된 총 실온 (RT) 200 μl / 잘 PBS/0.5 % 트윈 - 20와 함께 접시 4 번 씻으십시오. </li><li> 100 μl / 잘 PBS/0.05 % 트윈 - 5분의 20 %의 BSA와 RT에서 블록 1 시간. </li><li> 여전히 쌩쌩하게 거꾸로 판을 흔들어하여 차단 버퍼를 제거싱크대에. 커버 및 -20 ° C에서 접시를 저장하거나, 다음 단계로 바로 진행합니다. </li><li> PBS/0.05 % 회전에서 1 시간 동안 RT에서 품어 트윈 - 20 / 5% BSA, 50 잘 희석 / μl 1:1000의 볼륨 (또는 필요에 따라)에 1 ° 애비를 추가합니다. </li><li> RT 200 μl / 잘 PBS/0.5 % 트윈 - 20과 접시 세척기 10 분 결합된 총 접시 4 번 씻으십시오. </li><li> 기다려봐 퍼옥시데이즈 (HRP) 복합 2 · 애비, PBS/0.05 % 회전에 시간 RT에서 트윈 - 20 / 5% BSA에서 (또는 필요한) 1:5000 희석을 추가합니다. </li><li> 200 μl / 잘 PBS/0.5 % 트윈 - 20와 함께 접시 4 번 씻으십시오. 각 세차 플레이트 와셔와 RT에서 10 분 총입니다. </li><li> RT 10 분 각 잘하는 TMB 기판의 50 μl를 추가하여 번호판을 개발할 수 있습니다. 50 μl / 잘 SO 4 H 2 2N의를 추가하여 반응을 중지하고 접시 450 nm의를 참조하십시오. (팁 : 이전 읽는 absorbances에 우물에 기포를 분산하기 위해 플레이트 로터를 사용하여 2 분 1,500 rpm으로 원심 분리기). </li><li> 양적 및 통계 분석을위한 스프레드 시트 파일에 수치를 내보냅니다. </li></ol> 시약 <table border="1"><tbody><tr><td> PBS / butyrate </td></tr><tr><td> 135 MM NaCl </td></tr><tr><td> 2.5 MM KCl </td></tr><tr><td> 8 MM 나 2 HPO 4 </td></tr><tr><td> 1.5 MM KH 2 PO 4 </td></tr><tr><td> 10 MM 나 - butyrate </td></tr></tbody></table><table border="1"><tbody><tr><td> 용해 완충액 </td></tr><tr><td> 250 MM의 자당 </td></tr><tr><td> 10 MM 트리스 - HCL, pH를 7.4 </td></tr><tr><td> 10 MM 나 - butyrate </td></tr><tr><td> 4 MM MgCl 2 </td></tr><tr><td> 0.1 % 트리톤 X - 100 </td></tr></tbody></table><table border="1"><tbody><tr><td> 와시 버퍼 C </td></tr><tr><td> 250 MM의 자당 </td></tr><tr><td> 10 MM 트리스 - HCL, pH를 7.4 </td></tr><tr><td> 10 MM 나 - butyrate </td></tr><tr><td> 4 MM MgCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> 자당 쿠션 </td></tr><tr><td> 30 % (W / V) 씻어 버퍼 C에서 자당 </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal Nuclease 버퍼 </td></tr><tr><td> 5 MM 나포 4 버퍼, pH를 7.0 </td></tr><tr><td> 0.025 MM CaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> 코팅 버퍼 </td></tr><tr><td> 80ml 솔루션 170ml + 솔루션 B + 250ml DH 2 O </td></tr><tr><td> 솔루션 : 0.2 M 나 2 CO 3 </td></tr><tr><td> 솔루션 B : 0.2 M NaHCO 3 </td></tr></tbody></table> 5. 대표 결과 NU - 엘리사 방법, 염색질은 mononucleosomes의 형태로 준비로 로드할 수있는 준비가되어 몇 가지 동일한 접시 일련의 사용. 우리는 일반적으로 각각 다른 안티 - PTM 특정 항체 (ABS)로 탐색할 수 있도록 동일하게 - 로드된 접시의 시리즈를 준비합니다. 우리는 또한 항상 수정 상태 전체 염색질 로딩 컨트롤에 관계없이 histones을 감지 AB와 함께 탐색할 수있는 접시를 준비합니다. 이 컨트롤은 나중에 양적 다른 샘플에서 준비 nucleosomes의 PTM 내용을 비교하기 위해서는 필수적입니다. 우리가이 접근법을 사용하여 원시 흡광도 값을 수정 주어진 염색질 샘플 내에서 PTMS의 수준을 quantitate하려면 다음과 같이 첫째, 우리는 염색질을 (배경 일반적으로 무시할 수)가 포함된없는 컨트롤 우물에서 얻은 값을 사용하는 백그라운드 신호를 뺍니다. 우리는 그들의 수정 상태의 독립 nucleosomes을 감지 AB와 함께 assayed되었습니다 동일하게 읽어 접시에서 얻은 NU - 엘리사 결과에 PTM 특정 신호를 표준화. (우리는이 목적을 위해 histones H2A H2B 또는 특정 polyclonal 애비를 사용했습니다.) 그러면 염색질 각각의 희석에 대한 의미와 차이를 결정하고, 엘리사 분석의 선형 부분에서 얻은 데이터를 사용합니다. NU - 엘리사 수학 및 통계 분석의 상세한 예제는 이전에 다음 ID로 사보고되었습니다 <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="그림 1"/> 그림 1. NU - 엘리사 단계의 개략도 diagam. A. 포유 동물 세포 수확되며 B.의 핵은 다운스 H로 세포의 분리omogenization, C.는 세포 원심 분리 후 30 % W / V 자당 쿠션, D. 위에 로드된 중단, 핵은 펠릿로 입금되며 E, F. 염색질은 MNase에 의해 주로 mononucleomes로 현장에서 소화되며 G, H 전. 가용성식이 mononucleosomes는 hypotonic 치료 및 잔류 핵 물질의 반복 원심 분리에 의해 압축이 풀립니다. J.의 Mononucleosomes는 여러 샘플에서 (표시 샘프.이 경우에는 1, 2)는 동일하게 - 로드된 접시의 일련의 microtiter 우물에 코팅과 함께 심문 아르 총 염색질 로딩을 결정 PTM 특정 애비 및 PTM 독립 애비. 자신의 PTM 내용에 차이가 샘플 두개의 차동 신호 검출 수준의 K.의 묘사는 아직 같은 안티 H2B immunoreactivity로 판단, 유사한 전체 염색질의 콘텐츠를합니다.

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <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+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

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NU - 엘리사는 특정 세포 유형 내에서 현재 nucleosome PTMS의 글로벌 상태를 확인할 수있는 방법을 제공합니다. NU - 엘리사 연구는 글로벌 nucleosome 수정 상태가 분기하는 세포 유형 4 비교에서 다르다 것으로 나타났습니다. 세포가 같은 epigenetic modulatory 에이전트에 노출되면 또한, NU - 엘리사 PTM 프로필은 변경 trichostatin - A 때 또는 마우스 embyronic의 줄기 세포가 4 차별하고 있습니다. 방법은 또한 인간의 ES 세포 5 염색질 연구를 성공적으로 적용되었습니다. NU - 엘리사가 현재의 형태로, 세포 epigenome의 합계 내에서 현재 PTMS만을 합성 서명을 검색할 수 있습니다하는 것이 중요합니다. 이것은 특정 유전 loci에 걸쳐 특정 PTM의 분포를 확인할 수 있습니다 게놈 전체 염색질의 immunoprecipitation에서 현저하게 다릅니다. NU - 엘리사 절차의 ​​초기 단계는 포유류의 핵 6,7에서 mononucleosomes을 분리하기 위해 이전 방법에서 조정됩니다. 이러한 적응 방법은 포유 동물 세포에서 고품질 그대로 mononucleosomes을 얻기 위해 편법 방법을 제공하고, 그 결과 분수들은 염색질 콘텐츠에 포괄하지만, 추가 핵 물질의 큰 거래를 포함하고 있습니다. 특정 애비가 사용되므로, 비 nucleosomal 자료를 오염하는 것은 큰 순결을 요구 대량 사양 접근법과는 달리, NU - 엘리사 분석에 잘 용납입니다. 그러나, NU - 엘리사는 서쪽이 서부와 nucleosomal PTMS의 검출을위한 질량 분석기 좋은 대안을 제공함으로써, 4 모래 바닥보다 더 민감하고 양적입니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> Micrococcal Nucleae </td><td> 시그마 </td><td> N3755 </td><td> -20 ° C에 저장이 U/10μl 버퍼의 aliquots를 확인 </td></tr><tr><td> Nunc MaxiSorp Microtiter 플레이트 </td><td> 어부 </td><td> 12-565-135 </td><td> 접착제 플레이트 커버도 피셔를 통해 주문할 수 </td></tr><tr><td> 자동 플레이트 와셔 </td><td></td><td></td><td></td></tr><tr><td> 1 단계 TMB - 엘리사 기판 </td><td> 찌르다 </td><td> 34,028 </td><td> 4 스토어 ° C 사용하기 전에 RT 미리 따뜻하게 또는 사양서에 대한 감독 </td></tr><tr><td> Microtiter 플레이트 리더 </td><td> 바이오 래드 microplate 리더 (모델 : 680) </td><td></td><td> 대부분의 colorimetric 플레이트 판독기 모델 적합 </td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

eukaryotes의 게놈은 DNA와 단백질 모두를 포함 염색질로 존재합니다. 염색질의 기본 단위는 두 histones H2A, H2B, H3, H4 1 및 각과 관련된 DNA의 146 기본 쌍을 포함하는 nucleosome입니다. histones의 N - 터미널 꼬리는 리신과 아르기닌이 풍부하고 아세틸화, methylation 및 기타 사후 translational 수정 (PTMS)에 의해 게시 - transcriptionally 수정됩니다. nucleosomes의 PTM 구성함으로써 자연 2,3에서 epigenetic는 유전자 규정의 모드를 제공하고, 관련된 DNA의 transcriptional 활동에 영향을 줄 수 있습니다. 우리는 nucleosome 엘리사 (NU - 엘리사)가 양적 세포에서 추출한 nucleosomes의 글로벌 PTM 서명을 확인하기라는 방법을 개발했습니다. NU - 엘리사는 모래 바닥 서양보다 더 민감하고 양적이며, 특정 세포 유형의 epiproteomic 상태를 심문하는 데 유용합니다. 이 비디오 저널 기사는 NU - 엘리사 분석을 수행하는 자세한 절차를 보여줍니다.

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

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

Nucleosome 엘리사 (NU - 엘리사)는 기본, 손상 nucleosomes의 준비에서 사후 translational 수정의 글로벌 패턴을 감지하는 민감한과 양적 방법입니다. 이러한 수정 methylations, acetylations 및 특정 히스톤 아미노산 잔류물에 phosphorylations를 포함하고, 따라서 NU - 엘리사는 특정 세포 유형의 전반적인 염색질 수정 상태의 글로벌 proteomic 분석을 제공합니다.

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 ...

detection-post-translational-modifications-on-native-intact

Nanyang Technological University

Research

JoVE Journal

Biology

17.2K 조회수.  Stanford University. Nucleosome 엘리사 (NU - 엘리사)는 기본, 손상 nucleosomes의 준비에서 사후 translational 수정의 글로벌 패턴을 감지하는 민감한과 양적 방법입니다. 이러한 수정 methylations, acetylations 및 특정 히스톤 아미노산 잔류물에 phosphorylations를 포함하고, 따라서 NU - 엘리사는 특정 세포 유형의 전반적인 염색질 수정 상태의 글로벌 proteomic 분석을 제공합니다.

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

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Zymography의 기능 매트릭스 Metalloproteinases의 감지

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

연구

생물학

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

편광 상피 세포에 Plasmids의 Microinjection에 의해 작은 GTPases의 기능을 분석

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

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 ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

특정 게놈 지역과 enChIP으로 관련 분자의 식별의 분리

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

본래 동맥의 내피 층에서 산화 질소와 활성 산소의 욕실 얼굴 인식

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

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 ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

본래 식물을 표백제로 또는 추출물의 열처리에 의한 담배 숙주 세포 단백질 제거 방법의 비교

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...