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><tbody><tr><td>Petri Dishes</td><td>VWR</td><td>NC-10747</td><td>or comparable</td></tr><tr><td>Bacto yeast extract</td><td>Difco</td><td>0217-17</td><td></td></tr><tr><td>Bacto peptone</td><td>Difco</td><td>0118-17</td><td></td></tr><tr><td>Bacto agar</td><td>Difco</td><td>0140-01</td><td></td></tr><tr><td>Dextrose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0514</td><td></td></tr><tr><td>Galactose</td><td>Sigma</td><td>G0750</td><td></td></tr><tr><td>Sc-Ura drop out media</td><td>MP Bio</td><td>114410622</td><td></td></tr><tr><td>Small Culture tubes</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Large Erlenmeyer Flask</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Centrifuge JA-10 rotor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (500 ml)</td><td></td><td></td><td></td></tr><tr><td>Falcon tube (50 ml)</td><td>VWR/Fisher</td><td>21008-951</td><td></td></tr><tr><td>PBS Tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>FastPrep Tubes (2 ml screw cap tube)</td><td></td><td></td><td></td></tr><tr><td>FastPrep Machine</td><td>MP Biomedicals</td><td>116004500</td><td></td></tr><tr><td>Zircon Beads</td><td>BioSpec</td><td>11079105</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014&amp;nbsp;</td><td></td></tr><tr><td>Imidazole (pH = 7.4)</td><td>Sigma</td><td>I5513&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>93482</td><td></td></tr><tr><td>Complete Inhibitor Cocktail (EDTA Free)</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail 1</td><td>sigma</td><td>P2850</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379&amp;nbsp;</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A1852&amp;nbsp;</td><td></td></tr><tr><td>Shaking Incubator (30 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Nickel Affinity Resin</td><td>Life Technologies</td><td>R901-01</td><td></td></tr><tr><td>G25 column</td><td>GE life sciences</td><td>27-5325-01&amp;nbsp;</td><td></td></tr><tr><td>Nutator</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>SDS-PAGE Gel (NuPage)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Coomassie Blue Stain</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Monoclonal V5 Antibody</td><td>Life Technologies</td><td>R960-25</td><td></td></tr><tr><td>Alexa647 Tagged monoclonal V5 Antibody</td><td>Life Technologies</td><td>451098</td><td></td></tr><tr><td>Protein Microarrays Kit</td><td>Life Technologies</td><td>PAH0525013</td><td></td></tr><tr><td>Genepix Scanner</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Lifter Slips</td><td>Thomas Scientific</td><td></td><td>http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td></tr><tr><td>Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT,&lt;br/&gt; 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&amp;nbsp; (add just prior to use)</td><td></td><td></td><td>Add the reagents to 1x 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><td>Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td><td></td><td></td><td>Add the reagents to 1x PBS to obtain the desired volume of blocking buffer</td></tr><tr><td>Probe Buffer: 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl, 500 &amp;mu;M ATP (for kinases), 1% BSA</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 &amp;ndash; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td><td></td><td></td><td>Before you begin, it is important to note the concentration gradient of imidazole (50 &amp;ndash; 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 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>3x YEP +6% galactose: Dissolve 30 g of yeast extract and&amp;nbsp; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td><td></td><td></td><td>&amp;nbsp;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 ...

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

Research

JoVE Journal

Biology

8.0K 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

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

블루 기본 페이지와 단백질 상관 관계 프로파일 링에 의한 선호도 정제 단백질 복합체 해결

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

연구

생화학

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

세포 외 단백질 Microarray 기술은 낮은 친 화력 수용 체 Ligand 상호 작용의 높은 처리량 검색

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

호스트 병원체 단백질 - 단백질 상호 작용의 풍경의 특성을 비교 접근

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

면역학 및 감염병학

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

멀티플렉스 동시 면역 침전을 이용한 단백질 상호 작용 네트워크 역학의 정량화

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

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 ...

생물학

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC - 면역 정량 프로테오믹스를 사용하여 포유 동물 세포에있는 단백질 상호 작용 파트너의 식별

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

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 ...

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 ...

Protein-protein Interfaces

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide chain or string.
Various computational and biochemical methods are used to study protein interfaces. Laboratory methods, such as affinity purification, mass spectrometry, and protein microarrays, can be used to identify new interactions. Co-immunoprecipitation of proteins and yeast two-hybrid screening are widely used to provide evidence on whether two proteins interact in vitro.&#160; Computer programs can predict PPIs based on similar interactions found in other proteins by comparing protein sequences and three-dimensional structures. Other computational approaches, such as phylogenetic profiling, predict PPIs based on the coevolution of binding partners. Additionally, gene fusion analysis is used to predict interaction partners by finding protein pairs that are fused in the genome of other organisms.&#160;
Proteins typically interact with multiple partners either at the same or different times, and they may contain more than one interaction interface.&#160; Many proteins form large complexes that perform specific functions that can only be carried out by the complete complex. In some cases, these protein interactions are regulated; that is, a protein may interact with different partners based on cellular needs. Further computational and statistical analyses sort such interactions into networks, which are curated in online interactome databases. These searchable databases enable users to study specific protein interactions, as well as design drugs that can enhance or disrupt interactions at the interface.

Protein Complexes and Protein-Protein Interactions

단백질-단백질 접점

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs ...