우리는 중요한 실험 평가와 토론을 UIC에서 오브라이언 실험실 감사합니다. 임상 및 번역 과학 그랜트 UL1TR000050에 대한 UIC 센터 -이 작품은 번역 과학의 발전에 대한 GKC 및 국립 센터 미국 심장 협회 부여 11SDG5230003에 의해 지원되었다.

1. 세포 형질 <ol><li> 형질 전에 플레이트 세포는 하루 면역에 대한 적은 세포를 도금. <ol><li> 면역 경우 : 6 잘 플레이트, 코팅 유리 커버 전표에서 (1도 기준) 적어도 4 시간 동안 37 ° C에서 0.02 % 젤라틴으로, 중간에 한 번 씻고, 각 웰에 대한 접시, 1 × 10 5 HEK 293T 2 ㎖ 완전한 성장 배지에서 세포 [Dulbecco의 수정 이글의 미디어 (DMEM)에 10 % FBS]. </li><li> 유세포 및 서양 얼룩 분석을위한 : 판 1.2 5 HEK 293T 세포 / 잘 6 잘 플레이트 (24 - 웰 플레이트에 잘 당 0.5 ML 전체 성장 매체에서 0.3 X 10 5 HEK 293T 세포에 2 ㎖ 전체 성장 매체에서 x 10 ). </li></ol></li><li> 제조의 프로토콜에 따라 형질에 대한 DNA를 준비 Effectene는 (Qiagen의) 면역에 사용됩니다. 대중 교통 LT1 (마이 러스)는 유세포과 서양 얼룩 분석에 사용됩니다. 사용되는 DNA의 양이 아래에 나열되어 있습니다. <table border="1"><tbody><tr><tD&gt; </td><td> 24 - 웰 플레이트 (NG) </td><td> 6 잘 플레이트 (NG) </td></tr><tr><td> CFP - 벡터 </td><td> 10 </td><td> 50 </td></tr><tr><td> VN N-AKAP-LBC </td><td> 200 </td><td> 1000 </td></tr><tr><td> VC N-PDE4D3-FL (전체 길이 PDE4D3) </td><td> 2.5, 5, 10, 20, 40, 80 </td><td> 100 </td></tr><tr><td> VC N-PDE4D3-UCR1 (UCR1 도메인을 포함하는 PDE4D3 N-말단 단편) </td><td></td><td> 100 </td></tr><tr><td> VC N-PDE4D3-UCR2 + CAT (UCR2 및 촉매 도메인을 포함하는 PDE4D3 C-말단 단편) </td><td></td><td> 100 </td></tr></tbody></table> 형질 Effectene (6 자)를 사용하여 : <ol><li> 상온에서 5 분 동안 표시된 플라스미드 DNA의 양을 + 4 μL 향상제 100 μL 버퍼 EC로, 혼합 및 부화를 추가합니다. </li><li> 10 분 동안 5 μL Effectene, 혼합과 부화를 추가실내 온도. 추가 셀에 드롭 현명. </li></ol> : 대중 교통 LT1 (6-well/24-well)를 사용하여 형질 전환 <ol><li> 멸균 튜브 옵티-MEM I 혈청이없는 배지 250 μl/50 μL에 플라스미드 DNA의 지정된 금액을 추가합니다. </li><li> 희석 된 DNA 혼합물에 대중 교통 LT1 시약을 추가하고 섞어 부드럽게 피펫. (대중 교통 LT1 시약 : DNA 비율은 DNA 1 μg 당 대중 교통 LT1 시약 3 μL)입니다. </li><li> 실온에서 30 분 동안 배양 및 세포 드롭 현명한 추가 할 수 있습니다. </li></ol></li></ol> 2. 유동 세포 계측법, 서쪽 오점, 그리고 면역 형광에 대한 세포의 준비 <ol><li> 24 시간 게시물 형질 세포를 수확하기 전에 epifluorescence에 현미경으로 형광을 확인합니다. </li><li> 유세포 분석을위한 세포의 준비 : <ol><li> 24-well/6-well 플레이트 0.5 ML / 2 ML 얼음처럼 차가운 PBS로 세포를 씻으십시오. </li><li> 50 μl/250 μL 0.05 % 트립신을 사용하여 세포를 분리합니다. </li><li> 200 μL / 1 ML DMEM 매체와 트립신을 중화. </li><li> 공동5 분 500 XG에서 원심 분리하여 llect 250 μl의 세포 (모두 24 잘 6 잘 플레이트), 웨스턴 블롯 분석을위한 나머지 셀 (1 ML)을 사용합니다. </li><li> 250 μL FACS 버퍼에 세포를 resuspend을 [PBS + 0.5 % (W / V) BSA + 2 mM의 EDTA (에틸렌 다이아 민 테트라 초산)] 유세포 분석을위한 얼음, 저장. 유동 세포 계측법 분석을 즉시하지 않을 경우 전지도 (PBS)에 1 % 파라 포름 알데히드에 고정 할 수 있습니다. </li></ol></li><li> 서양 얼룩 분석을위한 세포의 준비 : 유동 세포 계측법 분석을위한 세포의 준비와 병행 실시했다. <ol><li> 2 ML 얼음처럼 차가운 PBS와 1X 세포를 씻으, 세포 용해 버퍼 100 μL [10 MM 나트륨 인산염 완충액, pH가 6.95, 150 mM의 NaCl을 5 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산), 5 mM의 EGTA, 1 % 트리톤 X-100]을 추가하여 세포를 용해 . </li><li> 4에서 21,000 XG에서 10 분 동안 원심 분리하여 세포 파쇄물을 수집 ° C. </li><li> , 브래드 포드 단백질 분석에 의해 단백질 농도를 정량화 SDS-PAGE 레인 당 단백질의 동등한 양을로드하고 immunoblotting을위한 니트로 셀룰로오스로 전송할 수 있습니다. </li></OL&gt; </li><li> 면역에 대한 세포의 준비. <ol><li> 2 ML PBS로 3 배 세포를 씻어 상온에서 10 분 동안 3.7 % 파라 포름 알데히드 (PBS)에 1 ㎖의 세포를 수정 후, 5 분 동안 2 ML PBS로 3 배 씻는다. </li><li> 장착 매체를 사용하여 유리 슬라이드에 슬립 커버 장착합니다. 필요한 경우 매니큐어를 사용하여 coverslip에 가장자리를 밀봉. </li><li> 시험에 ° C 이전 4시에 슬라이드를 저장합니다. </li></ol></li></ol> 3. 유동 세포 계측법 및 면역 형광 현미경 <ol><li> 유동 세포 계측법은 베크 만 보습 바로 앞에 달린 풀 베는 시안 488 nm의 장착 ADP, 405 nm의 및 642 nm의 고체 레이저를 사용하여 수행됩니다. 정상 회담 소프트웨어는 수집 및 분석에 사용됩니다. <ol><li> 표준 구슬을 사용하여 흐름 cytometer에를 보정합니다. </li><li> 게이트 라이브 셀 인구 리니어 스케일에 플롯 앞으로 분산 형 (FSC) / 옆으로 분산 (SSC). </li><li> 게이트 집계되지 않은 살아있는 세포 인구 플롯 전압 / FSC 펄스 폭. </li><li> 플롯 count/FL6 (CFP fluorescenc게이팅 비 집계 라이브 CFP-양성 세포에 대한 로그 스케일 E, 405 NM). </li><li> BiFC 강도에 대한 로그 스케일로 플롯 count/FL1 (YFP 형광, 488 nm의). </li><li> FSC, SSC, FL1과 FL6 사진 배율 튜브 (PMT) 각 신호에 대한 임계 값을 설정하는 전압을 조정, YFP - CFP 더블 부정적인 샘플을 실행합니다. </li><li> 실행 (두 YFP +와 CFP +)에 대한 샘플 향후 오프라인 보상을 양성 하나 </li><li> 분석을위한 최소 10,000 게이트 이벤트 (비 집계 라이브 CFP + 세포) 취득. </li></ol></li><li> . 면역 형광 현미경 자이스 혈구 LSM 510 공 촛점 현미경 참고를 사용하여 수행됩니다 : 그것은 모든 설정을 사이에 일관성 (예 : 줌, 핀홀, 검출기 이득 증폭기 오프셋, 프레임 크기, 스캔 속도, 스캔 평균 및 레이저 파워)를 유지하는 것이 중요합니다 모든 데이터의 적절한 비교를위한 샘플입니다. </li></ol> 4. 데이터 분석 (정상 소프트웨어를 사용하여 수행) <ol><li> acquisit에 대한 것과 비슷한 분석 프로토콜을 설정적절한 게이팅 이온 : 살아있는 세포에 대한 FSC / SSC 도트 플롯 문 1. 집계되지 않은 살아있는 세포에 대한 게이트 1 FSC / 펄스 폭 문 2. 비 집계 CFP + 라이브 세포 게이트 3 수 / CFP의 문 3. </li><li> YFP와 YFP +와 CFP + 싱글 양성 세포를 사용하여 CFP 신호를 보상한다. </li><li> CFP / YFP 양성 세포를 다시 확인 커트 오프 라인. </li><li> 수출 YFP는 CFP의 형광 강도 (MFI) + 데이터 플롯 Excel로 세포를 의미한다. </li><li> AKAP-LBC, PDE4D3의 발현에 YFP MFI을 정상화하고, 서양 얼룩에 의해 결정, α-tubulin에 컨트롤을로드. </li></ol>

<ol>
	<li>Collura, V., Boissy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+protein-protein+complexes+to+interactomics.">From protein-protein complexes to interactomics.</a> Subcell Biochem. 43, 135-183 (2007).</li><li>Stumpf, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).</li><li>Shekhawat, S. S., Ghosh, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-protein+systems:+beyond+binary+protein-protein+interactions.">Split-protein systems: beyond binary protein-protein interactions.</a> Curr. Opin. Chem. Biol. 15, 789-797 (2011).</li><li>Dwane, S., Kiely, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+used+to+study+how+protein+complexes+are+assembled+in+signaling+cascades.">Tools used to study how protein complexes are assembled in signaling cascades.</a> Bioeng. Bugs. 2, 247-259 (2011).</li><li>Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+measuring+protein+interactions+in+vivo+and+in+vitro.">New methodologies for measuring protein interactions in vivo and in vitro.</a> Curr. Opin. Struct. Biol. 15, 4-14 (2005).</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., Suarez, C. D., Hu, 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=Visualization+of+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nat. Protoc. 3, 1693-1702 (2008).</li><li>Wong, K. A., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation.">Bimolecular fluorescence complementation.</a> J. Vis. Exp. (50), e2643 (2011).</li><li>Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+general+principles+and+applications+to+selected+studies+in+tumor+biology.">Flow cytometry: general principles and applications to selected studies in tumor biology.</a> Int. Adv. Surg. Oncol. 2, 125-153 (1979).</li><li>Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+GLUT4+translocation+to+the+plasma+membrane+by+flow+cytometry.">Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry.</a> J. Vis. Exp. (45), e2429 (2010).</li><li>Smith, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+confocal+microscopy.">Basic confocal microscopy.</a> Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).</li><li>Westwick, J. K., Lamerdin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+discovery+with+contextual+assays+and+cellular+systems+analysis.">Improving drug discovery with contextual assays and cellular systems analysis.</a> Methods Mol. Biol. 756, 61-73 (2011).</li><li>Kilpatrick, L. E., Holliday, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+pharmacology+of+G+protein-coupled+receptor+signaling+complexes+using+bimolecular+fluorescence+complementation.">Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.</a> Methods Mol. Biol. 897, 109-138 (2012).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

BiFC 단백질 - 단백질 상호 작용을 연구하는 간단하고 민감한 방법입니다. 이 방법은 새로운 단백질 - 단백질 상호 작용을 식별하는 데 사용할 수 없습니다, 그러나, 그것은 세포 내부의 단백질 - 단백질 상호 작용을 확인하고 기능적 특성을 연구하는 것이 특히 편리하다; 같은 단백질 - 단백질 상호 작용 사이트의 매핑 단백질 복합체의 세포 내, 등, 그리고 단백질 - 단백질 상호 작용을 조절 수있는 ...

650,000 단백질 - 단백질 상호 작용은 정상 세포의 기능 1,2을 유지하는 중요한 역할을 놀고, 인간 interactome에 존재하는 것으로 추정된다. 공동 immunoprecipitation의 외에 (CO-IP), 금 표준 (PCA) 세포 안쪽으로 단백질 - 단백질 상호 작용 검출의 감도를 개선하기 위해 개발되었습니다, 세포 파쇄물에서 여러 단백질 조각 보완 분석 실험 단백질 - 단백질 상호 작용을 연구한다. 기술은 포스터 공명 에너지 전달 (FRET), 생물 발광 공명 에너지 전달 (브렛) 및 이분자 형광 보완 (BiFC) 4,5 (가) 있습니다. 각각의 잠재적 인 상호 작용 단백질 파트너 (이 예에서 우리는 AKAP-LBC 및 PDE4D3 사용)에 융합되어, BiFC는 형광 단백질의 두 조각 (YFP 변형 여기에서 우리는 비너스를 사용한다)의 촉진 협회를 기반으로합니다. F로 시각화 할 수 있습니다 기능 형광 세포 결과 안에 관심의 두 단백질의 상호 작용라이브 또는 고정 세포 6,7,8 중 하나를 사용하여 luorescence 현미경. 다른 PCA는 비교 BiFC는 살아있는 세포에서 세포 지방화를 공부하는 잠재력에 민감하고 덜 기술적 도전이다. 이 기술의 주요 단점은 있지만 한 번 형성된 형광 단백질 복합체가 반전 될 수 있다는 것입니다. 따라서 동적 단백질 - 단백질 상호 작용을 연구하는 좋은 방법은 아닙니다. 본 논문에서, 우리는 금성의 형광 강도를 모니터링하여 AKAP-LBC와 PDE4D3의 상호 작용 사이트를 매핑하는 BiFC 사용합니다. (자동 높은 처리량 기계를 사용하여 수행되지 않은 경우) 시간과 노동 집약적 형광 현미경을 사용하여 기존 분석에 비해 유동 세포 계측법은 짧은 시간 9을 통해 이기종 인구 세포의 수천의 간단한 정량 분석을 제공합니다 10. 여기 유세포-BiFC 분석 및 공동 IP 전통의 나란히 비교를 실시함으로써, 우리는 보여 그 이methods는 비교 데이터를 제공하지만, 유세포 BiFC 분석은 시간을 덜 소모하고 그 세포가 제한 될 때 더 유용 할 수 있습니다 적은 재료를 사용하고 있습니다.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's media (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin (pen/strep), 100x</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Flag antibody</td>
			<td>Sigma</td>
			<td>M2, F1804</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>16B12, MMS-101R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-tubulin</td>
			<td>Sigma</td>
			<td>DM1A, T9026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Clontech</td>
			<td>632569</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6-well tissue culture treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep plasmid purification kit, high speed</td>
			<td>Qiagen</td>
			<td>12663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-buffered saline (DPBS), sterile 1x</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05% (w/v)</td>
			<td>Gibco</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tubes for FACS staining</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>S74337MF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P-36931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superfrost plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisherfinest premium cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CO2 air-jacketed Incubator</td>
			<td>NuAIR DH autoflow</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope LSM510 META</td>
			<td>Carl Zeiss, Inc</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrophoresis and transfer unit</td>
			<td>Biorad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyan ADP Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

세포 내부의 단백질 - 단백질 상호 작용을 연구하는 방법 중, 이분자 형광 보완 (BiFC)은 비교적 간단하고 민감합니다. BiFC는 형광 단백질의 두 가지 비 형광 조각을 (금성, 노란색 형광 단백질의 변형은, 여기에 사용되는)를 사용하여 형광의 생산을 기반으로합니다. 비 형광 비너스 조각은 (VN 및 VC) 때문에 VN-AKAP-LBC-VC-PDE4D3 상호 작용과 기능 형광 단백질의 형성에 형광 항복, 두 개의 상호 작용하는 단백질 (이 경우, AKAP-LBC 및 PDE4D3)에 융합 세포 내부. BiFC 단백질 복합체의 세포 내 형광 강도에 따라 단백질 상호 작용의 강도에 대한 정보를 제공합니다. 그러나 단백질 - 단백질 상호 작용의 강도를 정량화하기 위해 현미경을 사용하여 BiFC 분석은 시간과 단백질 발현 및 상호 작용의 이질성으로 인해 다소 주관적이다. 유세포 흐름을 결합하여BiFC 방법론과 분석은 형광 BiFC 단백질 - 단백질 상호 작용 신호는 정확하게 짧은 시간에 세포의 대량을 위해 측정 할 수 있습니다. 여기, 우리는 AKAP-LBC와의 상호 작용에 필요한 PDE4D3의 영역을 매핑하기 위해이 방법의 응용 프로그램을 보여줍니다. 이러한 높은 처리량 방법은 단백질 - 단백질 상호 작용을 조절 심사 요소에 적용 할 수 있습니다.

AKAP-LBC 및 PDE4D3 구조 (그림 1B)는 각각 VN 및 VC 조각에 융합되었다. AKAP-LBC 및 기능 YFP 형광 (그림 1A)의 PDE4D3 결과의 상호 작용. 여기에서 우리는 PDE4D3의지도 AKAP-LBC-결합 부위에 BiFC 메서드를 사용합니다. 상류 보전 지역 1 (UCR1), 상류 보전 지역이 (UCR2)와 촉매 영역 (CAT) PDE4D3의 바인딩 사이트를 선별​​ 사용 하였다 따라서 전체 길이 (FL), UCR1 및 UCR2 플러스 CAT, PDE4 가족 단백?...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

이분자 형광 보완의 유세포 분석은 단백질 - 단백질 상호 작용을 연구하는 높은 처리량 정량 방법을 제공합니다. 이 방법은 매핑 단백질 결합 부위와 단백질 - 단백질 상호 작용을 조절하는 요인을 선별 적용 할 수 있습니다.

이분자 형광 보완의 유세포 분석 : 단백질 - 단백질 상호 작용을 연구하는 높은 처리량 정량 방법

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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Research

JoVE Journal

Biology

18.3K 조회수.  University of Illinois at Chicago. 이분자 형광 보완의 유세포 분석은 단백질 - 단백질 상호 작용을 연구하는 높은 처리량 정량 방법을 제공합니다. 이 방법은 매핑 단백질 결합 부위와 단백질 - 단백질 상호 작용을 조절하는 요인을 선별 적용 할 수 있습니다.

비디오: Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

연구

생물학

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

세계 S. cerevisiae의에서 organelle 형태를 연구하기 위해 높은 처리량 방법

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

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 ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

히타치 F - 7000 형광 분광 및 PicoGreen 염료를 사용하여 dsDNA의 부량

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Bimolecular 형광 Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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 ...

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 ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

고급 3D 형광 현미경을 사용하여 자식 작용의 정량 분석

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

담배 원형질체 및 잎에 이분자 형광 보완에 의해 시각 단백질 - 단백질 상호 작용

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...