저희 연구실의 작품은 VC에 NIH, 식품 및 농업의 국립 연구소 USDA, NSF, 바드,도 및 BSF로부터 기금을 지원합니다

1. 표현과 단백질의 추출 <ol><li> Differentially 자신의 검출을위한 테스트하는 단백질에 태그를 추가하십시오. 더 큰 크기 (예를 들어, GST)의 태그와 멤브레인 (ProIM)에 고정되어야하고, unfused 태그가 고정화 대조군 (ProIMnc)로 사용할 수있는 단백질을 분류. 하나 크거나 작은 태그와 가용성 프로브 (ProSOL)로 사용되는 단백질을 분류. 적절한 대조군 수용성 단백질 (ProSOLnc)에 동일한 태그를 융합하고 감지 바인딩의 특이성을 확인하기 위해 상호 작용 분석에 포함됩니다. </li><li> 태그 단백질을 표현할 수있는 단백질 표정 시스템, 즉, E.을 선택 대장균, baculovirus 등 단백질의 잠재적인 포스트 translational 수정 및 수율에 대한 요구 사항에 따라 다릅니다. </li><li> proteinase 억제제 칵테일을 포함한 SDS - PAGE 로딩 버퍼 (20 % 글리세롤, 4% SDS, 20 MM 트리스 - HCL, pH6.8)을 사용하여 선택의 유기체 (단계 1.2)에서 ProIM 및 ProIM NC의 압축을 풉니다. 15 분 상온에서 세포 현탁액을 남겨, 2 - 메르 캅 토 에탄올 5 분 끓여의 0.5 %를 추가합니다. </li><li> 표준 프로토콜 16을 사용하여 선택의 유기체 (단계 1.2)에서 ProSOL 및 ProSOL NC의 압축을 풉니다. 이러한 단백질은 단계 4.1에서 사용되는 (140 MM NaCl, 10 MM, 트리스 - HCL 산도 7.4, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 2 MM DTT, 1 % BSA, 0.1 % 트윈 20) 바인딩 버퍼에 쉽게 용해한다. </li><li> 이러한 바이오 래드 단백질 분석 키트와 같은 표준 방법을 사용하여 재조합 단백질의 농도를 결정합니다. 바이오 래드 단백질 분석 키트 등. 오히려 단백질을 정제보다 원유 추출물은, 분석을 위해 사용하는 경우, 쿠매시 브릴리언트 블루 R - 250과 SDS - polyacrylamide 젤 자국에 해당 밴드의 densitometry를 스캔하고 알려진를 사용하여이 추출에 대한 관심의 단백질의 농도를 추정 참고 BSA의 농도. </li></ol> 2. 멤브레인에 ProIM 및 ProIMnc의 고정 <ol><li> 추출물의 두 세트의 표준 프로토콜 16에 따라 SDS - polyacrylamide 젤에 ProIM 및 ProIM NC 1 μg를 포함하는 각를 해결합니다. </li><li> 전기 후, 전송 버퍼의 100 ML (60 MM 글리신, 10 MM 트리스, 0.0006 % SDS, 20 % MeOH)에에 전송에게 젤을 넣고 실온에서 20 분에 대한 부드러운 교반과 부화. </li><li> 표준 프로토콜에 따라 16 nitrocellulose 막에 겔에 포함된 단백질을 Electrotransfer. </li></ol> 3. 멤브레인 - 바운드 단백질 다시 접는 <ol><li> electrotransfer 후, 버퍼 15 ML에서 15 분 멤브레인을 품어 (30 MM 트리스 - HCL pH7.4, 0.05 % 트윈 20) 잔류 SDS를 제거하기 위해 부드러운 선동과 함께. </li><li> 신중하게 배수 후, 변성 버퍼 (7 M 구아니딘 염산염, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 50 MM DTT, 50 MM 트리스 - HCL pH는 8.3)의 25 ML에 멤브레인을 전송하기만하면됩니다. 부드러운 교반과 상온에서 2 시간 동안 부화. (참고 : 변성 버퍼에 incubated 때 nitrocellulose 막이 불투명됩니다.) 부드러운 교반과 상온에서 2 시간 동안 부화. </li><li> 25 TBS의 ML (10 MM 트리스 - HCL, 150 MM NaCl, 산도 7.4) 5 분 부드러운 교반과 부화 막을 전송 (참고 :이 단계 동안 멤브레인는 원래 흰 색을 찾으). </li><li> 바인딩 버퍼의 25 ML (단계 1.2 참조) 멤브레인를 전송 ° C 부드러운 교반과 함께 하루를 위해 4 알을 품다. </li></ol> 4. ProSOL하여 ProIM을 탐색 <ol><li> 이 스트립, 모두 포함 ProIM 및 ProIM NC에 멤브레인 컷. </li><li> 신선한 바인딩 버퍼의 10 ML에 ProSOL 또는 ProSOLnc 중 1-10 μg으로 준비를 diluting하여 ProSOL 및 ProSOLnc 하이브 리다이 제이션 솔루션을 확인하십시오. ProSOL 또는 ProSOLnc 하이브 리다이 제이션 솔루션으로 각 멤브레인를 전송하고 부드러운 교반과 함께 실온에서 1.5 H에 대해 숙고하다. </li><li> 하이브 리다이 제이션 솔루션에서 세포막을 제거하고 15 분 TBS의 각 세 번 씻어. </li></ol> 5. immunoblotting하여 단백질 - 단백질 상호 작용을 떠올리 <ol><li> 실온에서 1 H에 대한 TBST에 2.5 % 탈지 우유 (10 MM 트리스 - HCL, 140 MM NaCl, 0.05 % 트윈 20, 산도 7.4)와 멤브레인를 차단합니다. 제조 업체에서 권장하는 농도에서 TBST에 0.5 % 탈지 우유의 기본 항체 (안티 strepII polyclonal 토끼 항체)을 희석. </li><li> 항체 솔루션에서 차단 막을 장소, 4 실온에서 1 H 또는 숙박에 대해 incubated ° C 부드러운 선동과 함께. </li><li> 15 분 20 ML의 TBST에서 세포막을 린스, 두 번 부드러운 교반과 함께 실온에서 5 분. </li><li> 제조 업체에서 권장하는 농도에서 TBST에 0.5 % 탈지 우유의 기다려봐 퍼옥시데이즈 (HRP)와 복합 보조 항체를 (안티 - 토끼 IgG 항체) 희석. 보조 항체의 세포막을 플레이스솔루션과 부드러운 교반과 함께 실온에서 1 H에 대해 숙고하다. </li><li> 15 분 20 ML의 TBST에서 세포막을 린스, 두 번 부드러운 교반과 함께 실온에서 5 분. 최종 TBS에서 린스 후, HRP의 chemiluminescence 기판 (예를 들어, Millipore Immobilon 서부 chemiluminescent HRP 기판)를 사용하여 단백질 단백질 상호 작용을 시각화. </li><li> 로 5.4 단계 5.1에서 설명하는 적절한 보조 항체 다음 ProIM에 융합 태그에 대한 기본 항체와 같은 세포막을 프로브. 시각화 ProIM 및 ProIMnc는 단백질 막 오버레이 분석 (단계 5.5)에서 얻은 밴드의 신원을 확인하는 단계 5.5에서 설명했다. 이 단계에서 얻은 신호 단계 5.5에서 얻은 잔여 신호보다 훨씬 강력하기 때문에 대부분의 경우, 조직들이 필요하지 않습니다. </li></ol> 6. 대표 결과 : ANK - MP 상호 작용은 표피 세포 담배 (그림 1A)에 BiFC에 의해 관찰되었다. MP가 박테리아 또는 식물로 표현 매우 불용성 단백질이기 때문에 단백질 막 오버레이 분석은 체외 (그림 1B)에서이 상호 작용을 확인하기 위해 채택되었습니다. GST - MP (ProIM) 또는 unfused GST (ProIM NC) 1 μg을 포함하는 단백질 추출물은 nitrocellulose 막에 electrotransfer 다음 SDS - polyacrylamide 젤 전기 영동에 의해 해결되었다. 이러한 ProIMs가, 가용성 ANK - strepII (ProSOL), GST - MP하지만, unfused하지 GST와 탐지 구속력을 전시되었을 때 (그림 1B, 차선 1, 2, 차선 5, 6과 비교할 때). 또한, ProIMs의 동일한 세트가 추가로 ANK - MP 상호 작용의 특이성 (그림 1B를 보여주는 관련이없는 ProSOLnc 즉, strepII을 (NADH3 - strepII) 태그 Arabidopsis 세포질 NADH의 키나제, 아무 구속력이 관찰되지 아니와 읽혔어요 차선 3, 4). <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="그림 1"/> 그림 1. 생체내 및 시험 관내에서 TMV MP 담배 ANK의 구체적인 결합. 로 BiFC 감지 담배 표피 세포를 생활 (A) ANK - MP 상호 작용. MP와 ANK는 C - 터미널과 각각 YFP의 N - 말단 반쪽에 융합되면 강력한 YFP 신호들은 인코딩 유전자의 microbombardment 다음 담배 표피에 coexpressed 있었다 복원되었다. 이 BiFC 신호 plasmodesmata 15 진단하는 세포 주변에서 puncta에 축적. 체외에서 (B) ANK - MP 상호 작용으로 단백질 막 오버레이 분석에 의해 감지. GST - MP (ProIM) 또는 unfused GST (ProIMnc) 1 μg을 포함하는 단백질 추출물은 nitrocellulose 막에 electrotransfer 다음 15 % SDS - polyacrylamide 젤에 해결이되었습니다. GST - MPProIM과 GSTProIMnc는 ANK - strepII (ProSOL)의 0.5 μg / ML 함께 incubated했고, ANK 바인딩이에 복합 안티 - 토끼 IgG + M 이차 항체에 의해 다음, 안티 strepII 토끼 polyclonal 항체와 멤브레인을 탐색하여 검색되었습니다 HRP (레인스 1, 2). GST - MPProIM이나 GSTProIMnc 둘 다 strepII 태그 (ProSOLnc, 차선 3과 4)에 융합 관련이없는 단백질, Arabidopsis 세포질 NADH의 키나제와 교류. 이 분석에서 관찰 밴드의 정체성은 안티 GST 항체 (차선 5와 6)으로 막을 프로빙에 의해 확인되었다. GST - MP 및 GST contining 단백질 추출물에 포함된 미확인 단백질은 ANK - strepII와 반응하면서 멤브레인가 버퍼와​​ 함께 세탁하지 않고 변성 버퍼로 처리되었을 때, ANK에 GST - MP의 바인딩은 중요성을 보여주는, 손실됩니다 단계 3.1 전 변성 과정 (차선 7 및 8).

<ol>
	<li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9, 789-798 (2002).</li><li>Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+interacting+proteins+by+bimolecular+fluorescence+complementation+in+planta.">Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.</a> J Mol Biol. 362, 1120-1131 (2006).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+Caenorhabditis+elegans+using+bimolecular+fluorescence+complementation+analysis.">Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.</a> Nat Protoc. 3, 588-596 (2008).</li><li>Citovsky, V., Gafni, Y., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+protein-protein+interactions+by+bimolecular+fluorescence+complementation+in+planta.">Localizing protein-protein interactions by bimolecular fluorescence complementation in planta.</a> Methods. 45, 196-206 (2008).</li><li>Ohad, N., Shichrur, K., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+protein-protein+interactions+in+plants+by+bimolecular+fluorescence+complementation.">The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.</a> Plant Physiol. 145, 1090-1099 (2007).</li><li>Zamyatnin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+integral+membrane+protein+topology+in+living+cells.">Assessment of the integral membrane protein topology in living cells.</a> Plant J. 46, 145-154 (2006).</li><li>Citovsky, V., Knorr, D., Schuster, G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P30+movement+protein+of+tobacco+mosaic+virus+is+a+single-strand+nucleic+acid+binding+protein.">The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein.</a> Cell. 60, 637-647 (1990).</li><li>Heinlein, M., Epel, B. L., Padgett, H. S., Beachy, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+tobamovirus+movement+proteins+with+the+plant+cytoskeleton.">Interaction of tobamovirus movement proteins with the plant cytoskeleton.</a> Science. 270, 1983-1985 (1995).</li><li>Tomenius, K., Clapham, D., Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+by+immunogold+cytochemistry+of+the+virus+coded+30+K+protein+in+plasmodesmata+of+leaves+infected+with+tobacco+mosaic+virus.">Localization by immunogold cytochemistry of the virus coded 30 K protein in plasmodesmata of leaves infected with tobacco mosaic virus.</a> Virology. 160, 363-371 (1987).</li><li>Ding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+plasmodesmata+are+specific+sites+of+localization+of+the+tobacco+mosaic+virus+movement+protein+in+transgenic+tobacco+plants.">Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.</a> Plant Cell. 4, 915-928 (1992).</li><li>Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+30+kd+protein+of+tobacco+mosaic+virus:+involvement+in+cell-to-cell+movement+and+dispensability+for+replication.">Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication.</a> EMBO J. 6, 2557-2563 (1987).</li><li>Wolf, S., Deom, C. M., Beachy, R. N., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+protein+of+tobacco+mosaic+virus+modifies+plasmodesmatal+size+exclusion+limit.">Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.</a> Science. 246, 377-379 (1989).</li><li>Waigmann, E., Lucas, W. J., Citovsky, V., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+functional+assay+for+tobacco+mosaic+virus+cell-to-cell+movement+protein+and+identification+of+a+domain+involved+in+increasing+plasmodesmal+permeability.">Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.</a> Proc. Natl. Acad. Sci. USA. 91, 1433-1437 (1994).</li><li>Ueki, S., Spektor, R., Natale, D. M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANK,+a+host+cytoplasmic+receptor+for+the+Tobacco+mosaic+virus+cell-to-cell+movement+protein,+facilitates+intercellular+transport+through+plasmodesmata.">ANK, a host cytoplasmic receptor for the Tobacco mosaic virus cell-to-cell movement protein, facilitates intercellular transport through plasmodesmata.</a> PLoS Pathog. 6, e1001201-e1001201 (2010).</li><li>Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., Wingfield, P. T., Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2011).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP1,+an+Arabidopsis+protein+that+interacts+with+Agrobacterium+VirE2,+is+involved+in+VirE2+nuclear+import+and+Agrobacterium+infectivity.">VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.</a> EMBO J. 20, 3596-3607 (2001).</li><li>Chen, M. H., Sheng, J., Hind, G., Handa, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+the+tobacco+mosaic+virus+movement+protein+and+host+cell+pectin+methylesterases+is+required+for+viral+cell-to-cell+movement.">Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.</a> EMBO J. 19, 913-920 (2000).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이러한 접근 방식은 적어도 단백질의 바인딩을 버퍼에 쉽게 용해되는 중 하나가 성공적으로 단백질 17,18의 다른 조합에 적용했던 ​​단백질의 조합 간의 단백질 - 단백질 상호 작용을 테스트에 적합합니다. 이러한 조건 하에서 모두 불용성의 단백질 사이의 iInteractions이 프로토콜 테스트하실 수 없습니다. 또한, ProIM의 성공적인 refolding은 분석을 위해 중요합니다. 잔...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 단백질 분석 키트 </td><td> 바이오 래드 </td><td> 500-0001 </td></tr><tr><td> Proteinase 억제제 칵테일 </td><td> 시그마 </td><td> S8820 </td></tr><tr><td> 미니 프로 테우스 시스템 </td><td> 바이오 RAD </td><td> 165-8000 </td></tr><tr><td> 세미 드라이 서부 모래 바닥 SD electrotransfer 시스템 </td><td> 바이오 RAD </td><td> 170-3940 </td></tr><tr><td> 기다려봐 퍼옥시데이즈와 복합 안티 - 토끼 IgG 항체 </td><td> GenScript </td><td> A00098 </td></tr><tr><td> 안티 GST 토끼 polyclonal 항체 </td><td> GenScript </td><td> A00097 </td></tr><tr><td> 안티 strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> BioTrace, NT의 nitrocellulose 전송 막 </td><td> 팔 과학 </td><td> 27377-000 </td></tr><tr><td> Immobilon 서부 chemiluminescent HRP 기판 </td><td> Millipore </td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

서로 다른 단백질 간의 상호 작용 확인은 분자 수준에서 자신의 생물 학적 기능을 조사 중요합니다. 이 단백질 바인딩을 평가하는 시험 관내 및 생체내 모두에서 여러 가지 방법,, 그리고 서로의 단점을 보완 적어도 두 가지 방법이 신뢰할 수있는 통찰력을 얻기 위해 실시한다. 생체내 분석에 bimolecular 형광 complementation (BiFC) 분석은 살아있는 세포 내에서 단백질 단백질 상호 작용을 감지 수 가장 인기있는 최소 침습적인 접근 방식을 나타냅니다뿐만 아니라 상호 작용하는 단백질의 1,2의 세포 지방화를 확인하십시오. 이 분석에서는, GFP 또는 그 변종의 비 형광 N - 및 C - 터미널 반쪽이 두 융합 단백질 인해 테스트 단백질 &#39;상호 작용을 함께 가지고있을 때 테스트 단백질에 융합, 그리고, 형광 신호가 3-6 재구성합니다 . 그 신호가 epifluorescence이나 공촛점 현미경으로 쉽게 감지할 수 있기 때문에 BiFC은 살아있는 세포 3 단백질 단백질 상호 작용에 대한 연구를위한 세포 생물학 중에서 선택의 강력한 도구로 떠오르고있다. 이 분석은 그러나, 때로는 잘못된 긍정적인 결과를 생성할 수 있습니다. 예를 들어, 형광 신호가 오히려 그 때문에 특정 상호 작용 7, 작은 subcellular 구획의 포장 종료로 인해 서로 멀리로 7 나노미터을 배열이 GFP 조각에 의해 재구성 수 있습니다. 이러한 제한으로 인해, 라이브 셀 이미징 기술에서 얻은 결과는 단백질 상호 작용을 검출 다른 원칙에 따라 독립적인 방법으로 확인하여야한다. 공동 immunoprecipitation (CO - IP) 또는 글루타티온 transferase가 (GST) 풀다운 assays은 일반적으로 체외에서 단백질 - 단백질 상호 작용을 분석하는 데 사용되는 같은 대체 방법을 나타냅니다. 그러나 이러한 assays iIn 그러나, 테스트 단백질의 바인딩에 대한 반응 supportsused 버퍼에 쉽게 용해되어야합니다. 따라서 불용성 단백질을 포함한 구체적인 상호 작용은 이러한 방법에 의해 평가하실 수 없습니다. 여기, 우리는이 어려움을 circumvents 단백질 막 오버레이 바인딩 분석에 대한 프로토콜을 설명합니다. 단백질 중 하나가 막 매트릭스에 고정되기 때문에이 기법에서는, 가용성 및 불용성 단백질 간의 상호 작용을 안정적으로 테스트할 수 있습니다. 이 방법은 같은 BiFC 같은 생체내 실험에서와 함께, 용해와 불용성 단백질 간의 상호 작용을 충실하게 조사하고 특징에 대한 안정적인 접근을 제공합니다. 이 문서에서는 담배 모자이크 바이러스 (TMV) 사이 바이러스성 휴대 전화 전송 8-14 중 여러 기능을 미치는 운동 단백질 (MP), 그리고 최근에 확인된 식물 세포 interactor, 담배 ankyrin 반복 함유 단백질을 바인딩 (ANK ) 15,이 기술을 사용하여 증명합니다.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

시험 단백질 - 단백질 상호 작용이 단백질 기능의 해부를 위해 불가결이다. 여기서는 소개<em&gt; 체외에서</em가용성 단백질과 막 단백질 고정화 -을 알아내기 위해&gt; 단백질 단백질 결합 분석. 이 분석은 불용성 단백질 및 솔루션에서 단백질 간의 상호 작용을 테스트하는 신뢰성있는 방법을 제공합니다.

단백질 막 중첩 분석 : 가용성식이 및 불용성 단백질 간의 상호 작용을 테스트하는 프로토콜 체외에서</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

protein-membrane-overlay-assay-protocol-to-test-interaction-between

Research

JoVE Journal

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

22.0K 조회수.  State University of New York. 시험 단백질 - 단백질 상호 작용이 단백질 기능의 해부를 위해 불가결이다. 여기서는 소개 체외에서 단백질 단백질 결합 분석. 이 분석은 불용성 단백질 및 솔루션에서 단백질 간의 상호 작용을 테스트하는 신뢰성있는 방법을 제공합니다.

비디오: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

연구

생물학

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

헬리 우스 진 건을 사용하여 양파 세포에서 단백질 - 단백질 상호 작용에 대한 Bimolecular 형광 Complementation (BiFC) 어세이

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidic Mesophases의 막 단백질의 높은 처리량을 결정하기위한 로봇의 사용

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

체외 메틸화 분석에서 단백질 아르기닌 메틸화를 공부합니다

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

살아있는 세포의 단백질 조각 보완 분석 (PCA)에 의해 게놈 전체 단백질 - 단백질 상호 작용 상영

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase 보완성 이미징 분석 결과 Nicotiana benthamiana 잎 뚜렷이 결정 단백질 단백질 상호 작용 역동성에 대 한

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

BIFC-FRET-FLIM 분석체에 의한 MADS 박스 전사 계수의 두 단백제와 칼슘 센서 단백질 간의 삼자 상호 작용 결정

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

E. coli의 재조합 단백질 충전 소포의 생산 및 이미징

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

저자 스포트라이트: 재조합 단백질의 생산 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.