This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1. 프로브 준비 <ol><li> 문화 다음과 다른 단백질과의 상호 작용을 조사하기 위해 사용 V5 융합 키나제 프로브를 정제 : <ol><li> 갓 줄무늬 효모 균주 Y258을 사용 (마타 pep4-3, his4-580, ura3-53, leu2-3,112) (게이트웨이 벡터 pYES-DEST52에서 표현) V5 융합 단백질을 포함. 마이크로 어레이에 프로브로 분리 된 단백질을 사용합니다. 합성 완료 - 우라실 (SC-URA) / 2 % 덱 스트로스 / 한천의 효모 플레이트 및 냉동 문화 (-80 ° C 글리세롤 주)에서 3 일간 30 ℃에서 성장한다. </li><li> 하나의 식민지에서 스타터 문화 (5 ~ 20 ㎖)에 접종하고 30 ℃에서 플랫폼 (220-250 RPM) 또는 휠을 흔들어에 사우스 캐롤라이나 우라 / 2 % 포도당에서 하룻밤 성장. </li><li> 다음 날 아침, 최종 OD 0.1 600에 충분한 스타터 문화와 사우스 캐롤라이나 우라 / 2 %의 라 피노 오스 문화의 400 ml에 접종. </li><li> 0.6 (600)는 V5 키나아제 쿵푸의 갈락토스 유도 다음 OD하기 위해 접종 성장시온 그래서 갈락토스의 최종 농도가 2 % 인 (3)의 인자에 의해 유도 배지를 희석하기에 충분히 첨가하여, 6 % 갈락토스로 보충 3X 효모 추출물 / 펩톤 (YEP)의 용액을 첨가함으로써 발현을 구성. </li><li> 흔들리는 플랫폼에서 6 시간 동안 30 ° C에서 세포를 유도한다. 적절한 통풍을 보장하기 위해 2 L 삼각 플라스크를 사용합니다. </li><li> 4 ° C에서 5 분 동안 1,000 XG에 세포 현탁액 400ml를 회전하여 JA-10 (또는 유사한) 로터를 사용하여 세포를 수확. </li><li> 얼음 50 ml의 차가운 PBS는 버퍼와 50 ML 원뿔 튜브로 전송 한 번 세포를 씻으십시오. 용해를위한 튜브 캡 스냅 ㎖로 2 용해 및 전송에 사용 (세제 또는 기타 첨가제없이) 얼음 차가운 PBS 버퍼에 다시 펠렛을 씻으십시오. </li><li> 펠릿을 피펫 거리 버퍼 4 ° C에서 1 분간 20,000 XG에서 세포를 스핀. </li><li> 얼음에 튜브를 넣고 용해 단계를 진행합니다. </li><li> 를 Lyse 세포 (250-350 μL 펠렛) 0과1 : 1에서 0.5 mm의 지르코니아 비드를 세포 펠렛, 비드, 인산염 완충 식염수 (PBS)를 1 볼륨을 4 ℃에서 2 분 간격으로 버퍼와 소용돌이를 교반 플랫폼을 3 회 사용하여 혼합물을 용해. </li><li> 4 ℃에서 10 분 동안 탁상의 microfuge에 20,000 XG에 해물을 원심 분리기. </li><li> 피펫 폴리 카보네이트 고속 원심 분리기 튜브에 뜨는 4 ° C에서 15 만 XG에 30 분 동안 ulracentrufugation에 의해 명확하게. </li><li> 히스티딘을 캡처 4 ℃에서 2 시간 동안 nutator에 미리 세척 니켈 2 + 친 화성 수지 (~ 100 μL)를 포함하는 튜브에 명확히 해물을 전송하고 부화 (그의) 6X는 V5-융합 단백질 태그. </li><li> 세척 버퍼와 4 ℃에서 10 분 동안 수지 세 번 씻으십시오. 4 ° C에서 1,000 XG에서 5 분을위한 테이블 탑 원심 분리기를 사용하여 수지 펠렛과 뜨는을 대기음. 각 세척 신선한 세척 버퍼를 추가하고 세척하는 동안 튜브를 교반에 대한 nutator을 반환합니다. </li><li> 세척 인트 후PS이 완료, 용출에 대한 신선한 G-25 컬럼 세척 수지를 적용합니다. </li><li> 9 분획의 총 50 밀리미터 단위 500 mM의 이미 다졸을 100 mM의 이미 다졸로 시작 단계적인 방식으로 용출 완충액 25 μl를 적용한다. </li><li> 소듐 도데 실 설페이트 폴리 아크릴 아미드 겔을 사용하여 수집 된 분획을 분석 (SDS-PAGE) 및 분석에서의 사용을 위해 가장 고농축 분획 (들)을 식별하고 -80 ° C에서 30 %와 저장소에 글리세롤을 추가 쿠마시 염색에 사용까지 분석. </li></ol></li></ol> 2. 배열 프로브 참고 : 분석을 시작하기 전에 항체 용액을 제조하는이 섹션의 단계 2.6을 참조하십시오. <ol><li> 상호 작용을 검출하는 프로브 버퍼에 260 ng를 / ㎖로 V5-형광 물질 항체 (예 : AlexaFluor 647)를 희석 흔들어 잘 섞는다. 참고 : 사용하고 nutat에 튜브를 배치 30 분 이전에이 항체 솔루션을 준비또는 또는 휠 완전한 혼합 및 항체의 균질 한 현탁액을 보장합니다. </li><li> 5-500 μg의 / ml의 농도 범위 V5 융합 단백질 프로브를 희석. 주 : 프로브 버퍼를 사용하여 각각의 단백질 - 단백질 상호 작용 분석을 위해 최적화. 최적화 버퍼를 조사하기 위해 더 많은 프로브를 추가 포함한다. 일반적으로 10 μg의 / ml의 시작점으로 이용되고, 신호 강도에 기초하여 적절하게 조정. </li><li> 냉동 (-20 ℃)​​에서 단백질 마이크로 어레이를 제거하고 사용하기 직전에 냉장고에 4 ℃에 가져다. </li><li> 누출을 방지하기 위해 파라 필름으로 위쪽 micorarrays 단백질을 함유하는 슬라이드 홀더에 직접 블로킹 완충액 추가 커버. 4 ° C에서 무대에서 50 rpm으로 흔들어 1 시간 동안 버퍼를 차단하는 배열을 차단합니다. </li><li> 차단 한 후, 4 ℃로 냉각 가습 실에 배열을 전송, 직접 배열 표면에 희석 프로브의 90 μl를 추가합니다. 도착 오버레이1.5 시간 동안 4 ℃에서 가습 실에서 제기 리프터 슬립 AYS 및 정적 배양 (NO 떨고). </li><li> 세 50 ML 원뿔 튜브 프로브 버퍼에 1 분마다 배열하는 과정을 3 회 반복한다. 완전히 슬라이드를 봉투에 충분한 사전 냉장 프로브 버퍼를 포함하는 원뿔 튜브 슬라이드를 추가합니다. 리프터 슬립 부드럽게 단백질 마이크로 어레이 (이 어레이의 표면에 손상을 줄 수 있기를 강제로 해제하지 않음)의 미끄러하도록 허용합니다. </li><li> 바로 이전 제기 리프터 슬립 세척 (단계 2.5) 및 오버레이를 완료 한 후 배열에 직접 항체 용액을 적용합니다. 습윤 챔버에서 4 ° C에서 30 분 동안 인큐베이션 배열. </li><li> 실온에서 5 분 동안 원심 탁상 800 XG에 50 ㎖ 원뿔형 튜브에 동일한 워시 (프로브 버퍼에서 1 분 3 회) 전 단계로서, 스핀을 수행한다. 647 nm에서 배열을 스캔하기 전에 30 분 동안 어둠 속에서 슬라이드 홀더에 배열을 공기 - 건조. </li></ol>

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The authors declare that they have no competing financial interests.

프로토콜은 원래 생체 한 새로운 키나제 상호 작용 네트워크의 식별 결과 효모 단백질 키나제의 구별과 관련 제품군 간의 결합 활성을 비교하는 (85) 고유의 효모 단백질 키나제 V5 융합 단백질을 사용하여 수행 하였다 선보였다. 신흥 프로테옴 프로파일 링 기술로, 높은 처리량 (HTP) 펩타이드 라이브러리, 진핵 생물과 원핵 생물의 모델 생물 8,17에 의존 단백질 마이크?...

단백질 생화학 및 생체 내에서 결합 활성의 글로벌 분석을 수행 할 필요가 단백질 - 단백질 상호 작용 (프로톤 펌프 억제제) 및 전체 프로테옴 1,3-8의 번역 후 변형 프로파일에 대한 새로운 방법의 발전을 가져왔다. 단백질 마이크로 어레이는 항체 (10, 11)를 포함하는 4-6,8,9 전체 길이 기능성 단백질을 사용하여 기능성 단백질 마이크로 어레이, 또는 분석 단백질 마이크로 어레이로 제조된다. 이들은 12 광범위한 분석을 생화학 실험 실시에 필요한 다양한 조건을 용이하게하기 위해, 표면의 화학 다양한 현미경 슬라이드 상에 배열 된 단백질의 고밀도를 포함하도록 설계된다. 같은 다른 사람 (13) 사이의 친 화성이 부착 그의 - 태그 단백질과 글루타티온를 연결하는 니켈 킬레이트 슬라이드로 라이신 또는 선호도 부착 방법을 통해 화학 부착 니트로 셀룰로오스와 알데히드 표면 화학. </p&gt; 단백질 - 단백질 상호 작용을 검출하는 기능성 단백질 마이크로 어레이를 사용하면 고품질의 기능성 단백질 라이브러리 (14)에 대한 액세스를 필요로한다. S.을 cerevisiae의 높은 처리량 크로마토 그래피 정제 기술과 단백질 구조를 태그 높은 복사 친 화성의 페어링을 통해 이러한 라이브러리를 생산하는 의무가있다. 효모 게놈의 대부분은 서열화되어 거의 전체 프로테옴 정화용 높은 카피 플라스미드로부터 발현 될 수 있으며, 생화학 (12)를 분석한다. 단백질을 수득 및 384- 웰 포맷으로 배열되면, 이들은 빠른 병렬 다중 파라 생화학 및 생물 정보학적인 분석을 가능하게 심문 8,14-16 현미경 슬라이드 상에 인쇄된다. 단백질 마이크로 어레이는 다른 많은 응용들 중 단백질, 지질, 소분자, 핵산과 효소 분석법과의 상호 작용을 위해 사용되어왔다. 프로테옴의 표면에 단백질의 접근성배열은 다음과 같은 분석 검출 다른 유형의 면역 친 화성에 그들이 순종 할 플라즈몬 공명, 형광 및 다른 많은 기술을 표면. 또한,이 생체 내에서 수행하기 어려울 수있는 실험 조건의 미세 조정을 허용한다. 이 프로토콜의 목적은 단백질 - 단백질 상호 작용을 검출하는 기능성 단백질 마이크로 어레이의 적절한 사용을 설명한다. 이 애플리케이션은 관심 고순도 검체 (단백질)를 사용하여 단백질 결합 활동 높은 처리량 병렬 생화학 분석을 가능하게한다. C 말단 (말단 카복시) 관심 V5 미끼 융합 단백질 단백질 정제를 위해 최적화 된 효모 균주에서 높은 카피 플라스미드로부터 생성되는 태그. C 말단 태깅 전장 단백질 번역 한 것을 보장한다. 본 연구에 사용 된 단백질은 His6X 태그를 통해 니켈 친 화성 수지를 사용하여 정제한다 Tda1-V5 융합 단백질 키나아제이다. Tda1-V5 융합 CONSTRUCt는 분석법에 사용하기에 가장 고농축 분획을 용리 이미 다졸 구배를 사용하여 용출을 통해 직렬 정제한다.

<table border="0" cellpadding="0" cellspacing="0" width="855">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Petri Dishes</td>
			<td style="width:180px;">VWR</td>
			<td>NC-10747</td>
			<td>or comparable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto yeast extract</td>
			<td style="width:180px;">Difco</td>
			<td>0217-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto peptone</td>
			<td style="width:180px;">Difco</td>
			<td>0118-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:180px;">Difco</td>
			<td>0140-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dextrose</td>
			<td style="width:180px;">Sigma</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Raffinose</td>
			<td style="width:180px;">Sigma</td>
			<td>R0514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Galactose</td>
			<td style="width:180px;">Sigma</td>
			<td>G0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sc-Ura drop out media</td>
			<td style="width:180px;">MP Bio</td>
			<td>114410622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Small Culture tubes</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Large Erlenmeyer Flask</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge JA-10 rotor</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge tubes (500 mL)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Falcon tube (50 mL)</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PBS Tablets</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Tubes(2ml screw cap tube)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Machine</td>
			<td style="width:180px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">116004500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Zircon Beads</td>
			<td style="width:180px;">BioSpec</td>
			<td align="right" style="width:119px;">11079105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Triton X100</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DTT</td>
			<td style="width:180px;">Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">MgCl2</td>
			<td style="width:180px;">Sigma</td>
			<td>M8266&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">NaCl</td>
			<td style="width:180px;">Sigma</td>
			<td>S3014&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Imidazole (pH = 7.4)</td>
			<td style="width:180px;">Sigma</td>
			<td>I5513&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PMSF</td>
			<td style="width:180px;">Sigma</td>
			<td>93482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Complete Inhibitor Cocktail (EDTA Free)</td>
			<td style="width:180px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Phosphatase inhibitor cocktail 1</td>
			<td style="width:180px;">sigma</td>
			<td>P2850</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSA</td>
			<td style="width:180px;">Sigma</td>
			<td>A2153&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Tween-20</td>
			<td style="width:180px;">Sigma</td>
			<td>P1379&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ATP</td>
			<td style="width:180px;">Sigma</td>
			<td>A1852&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Shaking Incubator (30 &#176;C)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nickel Affinity Resin</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R901-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">G25 column</td>
			<td style="width:180px;">GE life sciences</td>
			<td>27-5325-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nutator</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">SDS-PAGE Gel (NuPage)</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Coomassie Blue Stain</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R960-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa647 Tagged monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>451098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Protein Microarrays Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td>PAH0525013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Genepix Scanner</td>
			<td style="width:180px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:267px;">Lifter Slips</td>
			<td style="width:180px;">Thomas Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:291px;">http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td>
		</tr>
		<tr height="124">
			<td height="124" style="height:124px;width:267px;">Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT, 
			2 mM MgCl2, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&#160; (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS to obtain the desired volume of blocking buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Probe Buffer: 2 mM MgCl2, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl,500 &#956;M ATP (for kinases), 1% BSA</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:267px;">Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl2</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:267px;">Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl2, 500 mM NaCl, 50 &#8211; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Before you begin, it is important to note the concentration gradient of imidazole (50 &#8211; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:267px;">3x YEP +6% galactose: Dissolve 30 g of yeast extract and&#160; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;galactose should not be autoclaved</td>
		</tr>
	</tbody>
</table>

probing high-density functional protein microarrays to detect protein-protein interactions

~ 4,200 재조합 효모 단백질을 함유하는 고밀도 기능성 단백질 마이크로 어레이는 판독에 대한 V5 에피토프 태그를 함유하는 친 화성 정제 효모 키나제 융합 단백질을 사용하여 키나아제 단백질 - 단백질 상호 작용을 조사한다. 정제 키나제 키나제 V5 융합체는 GAL 유도 성 프로모터 하에서 구축 플라스미드를 함유하는 형질 높은 사본 단백질 생산을 위해 최적화 효모 균주의 배양을 통해 얻어진다. 효모는 2 %의 갈락토오스와 유도 한 다음 6 시간 동안 중립 탄소원과 제한 배지에서 성장시킨다. 그 다음, 배양 물 수거되고 키나제 분석에 사용하기위한 고순도의 단백질 키나아제를 얻었다 표준 친화 크로마토 그래피 기술을 사용하여 정제한다. 정제 키나아제 분석을 위해 적절한 범위로 키나아제 완충액으로 희석하고 단백질 마이크로 어레이는 종래 단백질 마이크로 어레이와 하이브리드 화를 차단한다. 혼성화 후, 어레이는 단클론 V5의 antib로게나ODY은 키나제-V5 단백질에 의해 결합 단백질을 식별합니다. 마지막으로, 어레이는 표준 마이크로 어레이 스캐너를 사용하여 스캔되고, 데이터는 생체 내에서 검증을 위해 하류 단백질 상호 작용의 세트 높은 신뢰도를 결정하기 위해 하류 정보학 분석 1,2- 위해 추출된다.

단백질 - 단백질 상호 작용 활성 Tda1-V5 단백질 키나제 융합 약 4,200 고유 S. 함유 효모 기능성 단백질 마이크로 어레이에 대해 미끼 단백질로서 구성 평가하는 표준 칩 판독기를 사용하여 관찰 cerevisiae의 GST 융합 단백질. 제품, GenePix 소프트웨어 또한 심문은 다양한 강도의 이벤트를 바인딩의 다수를 한 것으로 밝혀졌습니다. 친화도는 목표 (V5 키나아제)에 결합 모노클 V5 항체 - 형광 물...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

거의 전체 (S)를 포함하는 단백질 마이크로 어레이를 사용하여 cerevisiae의 프로테옴은 병렬 단백질 - 단백질 상호 작용의 수천의 신속한 편견 심문 프로브된다. 이 방법은 단백질 - 소분자, 번역 후 수정 및 높은 처리량의 다른 분석을 위해 이용 될 수있다.

고밀도 기능성 단백질 마이크로 어레이를 프로빙하는 단백질 - 단백질 상호 작용을 검출하기

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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Biology

7.9K 조회수.  Stanford University.  거의 전체 (S)를 포함하는 단백질 마이크로 어레이를 사용하여 cerevisiae의 프로테옴은 병렬 단백질 - 단백질 상호 작용의 수천의 신속한 편견 심문 프로브된다. 이 방법은 단백질 - 소분자, 번역 후 수정 및 높은 처리량의 다른 분석을 위해 이용 될 수있다. 

비디오: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

분할 Ubiquitin 기반 막 효모 두 하이브리드 (신화) 시스템 : 단백질 단백질 상호 작용을 확인을위한 강력한 도구

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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생물학

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

마이크로 패턴의 표면에 단백질 - 단백질 상호 작용에 - 생체내 감지

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

이미징 단백질 - 단백질 상호 작용 생체내에</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

을 바탕으로 단백질 - 단백질 상호 작용의 확인에 프로토콜<sup&gt; 15</sup&gt; N 신진 대사 라벨링, Immunoprecipitation, 수량 질량 분석법 및 선호도 변조

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

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 ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

화학적 매개 종간의 상호 작용을 검출하는 공 배양을 사용하여

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

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 ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

를 통해 교체 단백질 - 단백질 상호 작용의 억제제의 개발 : 설계 및 개발 비 ATP 경쟁 CDK 억제제에 응용

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...