국립 보건원 / NIDA의 DA034783 지원. 우리는 다량 체-PAGE와 기술 지원 브라이언 A 킬링거합니다.

1. 조직 준비 <ol><li> 200 x BN-PAGE 샘플 버퍼 (200 mM Bis (2- 하이드 록시 에틸) 아미노 - 트리스 (하이드 록시 메틸) 메탄 (Bis-Tris), 200 mM NaCl, 40 % w / v 글리세롤, 0.004 % Ponceau S, pH 7.2 ). 비고 :이 저장 용액은 미리 제조하여 4 ℃에서 보관할 수있다. </li><li> 1x 상업용 프로 테아 제 억제제 칵테일을 포함하는 750 μL dH 2 O에서 4x 샘플 버퍼 250 μL를 희석하십시오. 소용돌이 치고 얼음 위에서 식히십시오. </li><li> 30 ML 획기적인 깨끗한 dounce homogenizer와 1 ML 1X 얼음 차가운 BN - 페이지 샘플 버퍼에 대상 조직 20 MG을 균질. 참고 :이 시범 실험을 위해 대상 조직은 전체 쥐 뇌 조직입니다. 균질화 후 샘플을 2 % digitonin과 같은 중성 세제로 처리하여 막 단백질의 가용화 및 전기 영동을 허용 할 수 있습니다. </li><li> 1.5 ML의 microcentrifuge 튜브에 homogenate를 전송하고, 불용성 세포 내용을 펠렛에 30 분 14,000 XG에서 원심 분리기. 애프터R 원심 분리는 얼음에 깨끗한 튜브에 뜨는을 가만히 따르다. </li><li> bicinchoninic 산 (BCA) 또는 유사한 단백질 정량 분석법을 이용하여 상등액의 단백질 농도를 측정하기 위해 제조 지시에 따라. </li><li> 균질 시료 (S)에 세제를 포함 할 경우, 0.25 % 쿠마시에 균질 액을 가지고 충분한 수용액에 5 % 쿠마시 블루 G-250의 양을 추가한다. </li></ol> 2. BN-PAGE <ol><li> 25 mL의 20 배 청색 기본 희석 (BN)이로 버퍼 스톡 (1.0 M 비스 - 트리스, 1.0 M 트리 신, pH를 6.8) 전기 영동 475 mL의 DH 1X 실행 완충액 500 mL로 0.002 % 쿠마시 블루를 함유하는 2 O. <ol><li> 샘플 세제를 포함하는 경우, 그 대신 0.02 %를 함유하는 쿠마 1X 실행 완충액 250 mL의 어느 것도 포함하지 않는 다른 250 mL로 만든다. </li></ol></li><li> 4 ℃로 냉각 완충액 (들). </li><li> 청소 및 제조업체의 지침에 따라 카세트를 붓는 폴리 아크릴 아마이드 젤을 조립. </li><li&gt; 표준 제조법 7 항 BN 폴리 아크릴 아미드 겔 (3 % T 적층 층, 6 % T 해결 층)을 붓고 잘 빗 놓는다. </li><li> 겔을 중합 후에 DH 2 O와 카세트 헹구고 제조사의 지시에 따라 전기 영동 장치를 조립한다. </li><li> 잘 빗를 제거하고 1 배 BN-PAGE 실행 버퍼로 우물을 입력합니다. 샘플이 로딩 될 때까지 버퍼에 전체 내부 챔버를 채우기 피한다. <ol><li> 샘플 세제를 포함하는 경우, 0.02 % 코마시 블루를 함유하는 완충액으로 각 웰을 채운다. 참고 : 4 ° C에서 단계 2.7-2.9를 수행합니다. </li></ol></li><li> 겔 로딩 정보를 사용하여, 목적하는 웰에 20 μg의 단백질을 함유하는 균질 부피 피펫. 미사용 웰로 1X BN-PAGE 샘플 버퍼의 등가 부피 피펫. 참고 :에 추가 균질의 적절한 볼륨을 계산하기 위해 BCA 분석에서 결정된 단백질 농도를 사용하여우물. </li><li> 샘플이 로딩 된 후, 1X BN-PAGE 주행 완충액 내부 챔버를 채운다. 젤이 완전히 버퍼에 빠져들되어 있는지 확인합니다. 다음에, 제조자에 의해 지시 된 레벨로 1X 주행 완충액 외부 챔버를 채운다. <ol><li> 샘플 세제를 포함하는 경우, 0.02 % 코마시 블루를 함유하는 1X 버퍼, 염료 무 1X 런닝 버퍼와 외부 챔버와 내부 챔버를 채운다. </li></ol></li><li> 전원 공급 장치에 전극을 연결하고, 상기 염료 밴드 층으로 해결 ~ 2cm까지 진행됨에 150 V의 겔의 단백질 electrophorese. 중지하고 전원 공급 장치를 분리합니다. </li></ol> 3. 교차 연결 참고 : 4 ° C에서 단계를 3.1-3.6를 수행합니다. <ol><li> 전기 영동 장치를 분해 및 겔 카세트의 유리창을 분리한다. 제거하고 스태킹 층을 버린다. </li><li> 조심 다만 염료 전면의 하단 아래에 겔을 잘라. 갖다이 컷은 같은 부드럽고 똑바로 가능하도록 배려하고 젤의 사용하지 않는 부분을 폐기합니다. 이는 균질 샘플의 모든 단백질을 포함 폭 ~ 2cm, 폴리 아크릴 아마이드 젤의 수평 스트립을 떠날 것이다. </li><li> 겔 스트립의 가장자리에 따라 사용되지 않은 부분을 잘라. </li><li> 조심스럽게 작은 용기에 10 ㎖의 인산염 완충 식염수 (PBS)로 스트립을 배치하고 부드럽게 평형 30 분간 장동 섞는다. </li><li> 평형화 후 폐기하고 다른 10 ㎖ PBS로 대체. 피펫 500 μL의 25 mM의 DSP는 PBS로 디메틸 술폭 시드에 용해하고, 30 분 동안 상기와 같이 (단계 34) 계속 혼합한다. </li><li> 는 DSP 솔루션을 붓고. DSP 미 반응을 종결 2 % 소듐 도데 실 설페이트 (SDS)를 포함 0.375 M 트리스 (히드 록시 메틸) 아미노 메탄 히드로 클로라이드 (트리스 - 염산)의 pH 8.8, 10 ㎖의 추가. 15 분 동안 장동를 계속합니다. </li></ol> 4. SDS-PAGE <ol><li> 겔 스트립 급랭되지만, SDS-PA를 제조표준 방법에 따라 9 GE 겔 용액. 중합 시약을 추가하지 마십시오. </li><li> 급냉 후, 실온 ΒΝ 겔 스트립 돌아가서 새로운 카세트로 겔 스트립 캐스팅. <ol><li> 이렇게하려면 신중 젤을 선택하고 깨끗한 젤 카세트 스페이서 플레이트에 배치합니다. </li><li> 오리엔트 염료 앞의 바닥 즉, BN-PAGE 동안에 가장 멀리 이동 측이 새로운 카세트의 상단에 가까운되어야 새로운 카세트 상부 (가장 가깝도록 스트립; 겔 스트립이 종래로부터 대칭되어야 정위). 그림 3을 참조하십시오. </li><li> 상단 모서리에도 상기 커버 플레이트의 상부 에지가되는 위치에 놓여 지도록 배치 스트립 (즉, 그것은 새 겔의 정상에있을 것이다). 염료 전면 유리판의 수평 에지에 평행 확인. </li><li> t에 공간을두고 이격 벽 중 하나에 대해 절개 띠의 한쪽을 눌러젤 그 반대편 부어하고 단백질 표준 또는 사다리를로드한다. </li><li> 겔 스트립의 하단은 어떤 고르지 않거나 고르지 영역을 포함하는 경우, 신중하게 버려야. 존재하는 경우, 그들은 겔 중에 트랩 기포 주입한다. </li><li> 겔 스트립이 정확하게 위치되면, 스페이서 플레이트 위에 커버 플레이트를하다. 어떤 갇힌 공기 방울을 밀어 부드러운 압력을 적용합니다. </li><li> 제조업체의 지침에 따라 젤 주입 장치를 조립하기 위해 계속합니다. </li></ol></li><li> 분해능 겔 버퍼 중합 시약을 추가하고 혈청 피펫을 사용하여 제조 된 겔 카세트로 붓는다. 스택 층을위한 공간을 떠나, 절제된 BN-PAGE 젤 스트립 아래 ~ 2cm로 겔 카세트를 채 웁니다. </li><li> 부어 겔의 정상에 100 μL 부탄올을 추가하고 해결 층의 중합을 30 분을 허용한다. 부탄올을 붓고. </li><li> 스태킹 겔 용액에 중합 시약을 추가합니다. serolog 사용피펫의 iCal 겔 카세트 나머지 빈 공간을 채우도록 적층 층을 붓는다. <ol><li> 이 겔 스트립 아래의 공간을 채우고, 기포가 트랩되지 않도록 적층 층 붓고 같이 겔 카세트 틸트. </li><li> 스태킹 겔 버퍼 겔 스트립 아래의 빈 공간을 채우는 같이 서서히 레벨에 기초 겔 카세트를 반환한다. </li><li> 거의 오버 플로우 할 때까지 옆에 스태킹 겔 버퍼로 적출 된 젤 스트립에 빈 공간을 채우기 위해 계속합니다. </li></ol></li><li> 적층 층을 30 분 동안 중합하도록 허용. 주 : 겔 중합 동안 10 배 SDS-PAGE에서 러닝 완충액 (250 개 mM의 트리스 (히드 록시 메틸) 아미노 메탄 (트리스), 1.9 M 글리신, 1 % SDS)을 확인. 이것은 또한 미리 만들어 실온에서 저장 될 수있다. </li><li> 1X 작업 버퍼를 만들기 위해 450 mL의 DH 2 O에 50 mL의 10 배 SDS-PAGE 실행 버퍼 주식을 희석. </li><li> 적층 층을 중합 한 후, 겔로부터 주입 카세트 삭제장치는 DH (2) O로 세정하고, 제조자의 지시에 따라 전기 영동 장치를 조립한다. </li><li> 그 제조자에 의해 지정된 레벨로 외부 챔버를 채우 1X SDS-PAGE에서 러닝 완충액으로 완전히 내부 챔버를 채운다. </li><li> 다음 분자량 래더 또는 적절한 단백질 표준 적출 겔 스트립 공간로드. </li><li> 전원 공급 장치에 전극을 부착하고, 쿠마시 염색이 겔을 실행하면 (120)에서 V. 샘플 electrophorese 중지하고 전원을 분리. </li><li> (10) 결합 항체 및 단백질 electroblotting 검출의 표준 방법을 사용하여 겔 분석. </li></ol>

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저자가 공개하는 게 없다.

단백질 - 단백질 상호 작용은 생물이 수행하는 모든 작업에 중요하다. 이 때문에, 그들은 강렬한 조사 및 연구의 주제이다. 다량 체-PAGE는 캡처 분리 및 단백질 복합체의 다양한 분석을위한 신규 한 방법이다. 우리는 이전에 질병 관련 단백질의 α-synuclein의 11 oligimerization 연구에의 적용 가능성을 증명하고있다. 그림 2에서 설명하지만, 그것은 많은 단백질 복합체로 확...

<html> 정상 세포 기능은 단백질 - 단백질 상호 작용 1, 2에 따라 달라집니다. 그 결과, 인간의 질병은 종종 다양한 단백질 복합체 (3)의 조립 및 행동 교란에 의해 표시된다. 이러한 상호 작용의 특성을 할 수있는 능력 때문에 중요하다. 이러한 상호 작용을 검출하는 수단은 현재 자주 상호 작용 파트너 풀다운 하였다 목적 단백질의 정제를 필요로한다. 통상적 인 정제를 차동 원심 분리, 침전, 및 / 또는 크로마토 그래피 (4)에 의해 달성된다. 이 방법은 시간이 많이 소요되어 각 대상 단백질을 변경해야합니다, 종종 낮은 수율을 초래한다. 현대 정제 방법은 포획 분자 (5)에 결합 된 비드 로케이션 컬럼에 면역 또는 추출하여 단백질을 표적으로하는 펩티드 태그의 융합을 포함이것은 많은 단백질 신성이지만 &quot;&gt; 6. 그것은 잠재적으로 보통 결합 파트너에 대한 친 화성이 다른 구조의 결과, 타겟의 순서 변경을 필요로한다. 일부 단백질 - 단백질 상호 작용에 민감한 성질은이 방법이 적용되지 않을 수 있음을 의미 많은 시나리오. 또한, 단백질 - 단백질 상호 작용을 매핑하는 데 사용 풀다운 분석으로 인해 자유와 미끼 단백질의 비 네이티브 수준의 제한된 도로, 정확한 세포 사진을 캡처 할 수 있습니다. 이상적으로, 단백질 복합체가 정화 또는 풀다운 할 필요없이, 그 나라의 상태를 검출 할 수있다. 블루 천연 폴리 아크릴 아마이드 겔 전기 영동법 (BN-PAGE)의 나트륨 도데 실 설페이트 폴리 아크릴 아마이드 겔 전기 영동법 (SDS-PAGE)에 덜 변성 대안으로 개발 및 생체 시료 7의 일부 단백질 복합체의 분리를 허용 하였다. 그러나, BN-PAGE 단백질은 LAR에 기초하여 구분하기다른 분자 크기, 전하, 입체 구조, 및 결합을 포함한 변수의 GE 번호. 이러한 요소의 상호 작용은 종종 단백질의 공동 분리, 집계 형성, 가난한 단백질 밴드 해상도를 초래한다. 두 차원 네이티브 폴리 아크릴 아미드 겔 전기 영동은 이러한 문제 8의 모든 일부를 해결 있지만. 기본 분리와 관련된 합병증을 피하기 위해, 일부 저자는 조직 해물 4 단백질 복합체를 캡처하는 등의 디티 오비스 (숙신 이미 딜 프로 피오 네이트)와 같은 아민 반응성 가교 시약 (DSP)를 사용합니다. 이 처리 해물을 다음 변성 및 단백질 복합체의 원래 크기와 메이크업을 유지하면서, SDS-PAGE에 의해 분리 될 수있다. 가교 시약이 조직 용해 액에 다른 한 분자의 근접성, 그리고 단백질을 기반으로 반응하기 때문에, 많은 자유도를 가지고 확률 적, 비특이적 배경 크로스를 상호 작용할 수 있습니다-linking 특히 농축 샘플에서 높을 수있다. 이 어려운-해석 결과로 이어질 수 있습니다. 여기서는 하이브리드 BN-PAGE / SDS-PAGE 법의 사용을 입증 분리 복잡한 혼합물에 단백질 어셈블리를 감지하는 멀티 머 - 폴리 아크릴 아미드 겔 전기 영동 (다량 체-PAGE)을 불린다. 우선, 세포 용 해물을 통해 BN-PAGE 폴리 아크릴 아미드 겔에 현탁시킨다. 용 해물을 함유하는 겔은 가교 시약 DSP와 반응시킨다. 의사 고정화 약간 겔상에서 분리 된 단백질은 배경 가교제의 반응성이 감소 의미 훨씬 비특이적 반응 할 가능성이있다. 가교 후, 겔 밴드를 절제하고 SDS-PAGE로 분리 하였다. 생성 된 겔은 전형적 폴리 아크릴 아마이드 겔 전기 영동법과 연관된 임의의 수단에 의해 분석 될 수있다. 이 방법은 추가로 정제 또는 당기 다우 없이도 변성 조직 파쇄물에서 천연 단백질 복합체의 분리 및 검출을 허용엔.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;epsilon;-Aminocaproic acid</td><td>Sigma</td><td>A2504</td><td></td></tr><tr><td>Acrylamide</td><td>Acros Organics</td><td>164855000</td><td>Toxic.</td></tr><tr><td>Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td><td>BioRad</td><td>161-0148</td><td>Toxic.</td></tr><tr><td>Ammonium persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>Anti rabbit IgG-HRP from goat</td><td>Santa Cruz Biotechnology</td><td>sc-2004</td><td></td></tr><tr><td>Bicinchoninic acid assay kit</td><td>Thermofisher</td><td>23225</td><td></td></tr><tr><td>Bis-tris</td><td>Sigma</td><td>B9754</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Butanol</td><td>Fisher Scientific</td><td>A399-1</td><td></td></tr><tr><td>Chemiluminescence substrate kit</td><td>ThermoFisher</td><td>24078</td><td></td></tr><tr><td>Coomassie blue G-250</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Toxic.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>D128-1</td><td></td></tr><tr><td>Dithiobis(succinimidylpropionate)</td><td>Thermo Scientific</td><td>22585</td><td></td></tr><tr><td>Dry nonfat milk</td><td>LabScientific</td><td>M0841</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>Thermofisher</td><td>78430</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144SI-212</td><td>For titration. Caustic.</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-4</td><td>For PVDF membrane activation. Toxic.</td></tr><tr><td>Monoclonal anti &amp;alpha;-synuclein IgG from rabbit</td><td>Santa Cruz Biotechnology</td><td>sc-7011-R</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-tetramethylethylenediamine</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>N,N&#039;-methylenebisacrylamide</td><td>Acros Organics</td><td>16479</td><td>Toxic.</td></tr><tr><td>NP40</td><td>Boston Bioproducts</td><td>P-872</td><td></td></tr><tr><td>Polysorbate 20 (tween-20)</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Polyvinylidene fluoride transfer membranes</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217-3</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Thermo Scientific</td><td>88265</td><td></td></tr><tr><td>SDS solution (10% w/v)</td><td>BioRad</td><td>161-0416</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma</td><td>L37771</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Fisher Scientific</td><td>BP329-1</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-500</td><td></td></tr><tr><td>Tris-HCl (0.5 M buffer, pH 6.8)</td><td>BioRad</td><td>161-0799</td><td></td></tr><tr><td>Tris-HCl (1.5 M buffer, pH 8.8)</td><td>BioRad</td><td>161-0798</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE Imagequant LAS 4000</td><td>GE Healthcare</td><td>28-9558-10</td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td></td></tr><tr><td>Synergy H1 microplate reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Gel Former + Stand</td><td>Biorad</td><td></td><td></td></tr><tr><td>Microfuge 22R centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>2 mL dounce homogenizer</td><td></td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Ultrasonic tissue homogenizer</td><td>Fisher Scientific</td><td>FB120220</td><td></td></tr></tbody></table>

multimer-page: a method for capturing and resolving protein complexes in biological samples

정화 및 단일 단백질과 펩티드를 연구하는 많은 잘 개발 된 방법이 있습니다. 그러나, 대부분의 세포 기능들은 그들의 비공유 쉽게 정제 기술에 의해 교란 바인딩 때문에 조사하기 어려운 작용 단백질 복합체의 네트워크에 의해 수행된다. 이 작업은 안정화 이차원 폴리 아크릴 아미드 겔 전기 영동을 사용하여 개질 된 조직으로부터 천연 단백질 복합체를 분리하는 방법을 설명한다. 조직 파쇄 액은 단백질 겔로 짧은 거리를 마이그레이션 될 때까지 전류를인가하고, 비 변성 청색 기본 폴리 아크릴 아미드 겔 상에 로딩된다. 이주 된 단백질을 함유하는 겔 조각은 절제하고 공유 단백질 복합체를 안정화 아민 반응성 가교 시약 디티 오비스 (숙신 이미 딜 프로 피오 네이트)와 함께 배양된다. 가교 결합 된 복합체를 포함하는 겔 스트립이어서 소듐 도데 실 설페이트 폴리 아크릴 아미드 겔에 캐스팅되고, t그는 단지가 완전히 분리된다. 이 방법은 저렴하고 쉽게 배울 수 의미, 기술과 대부분의 분자 생물 학자들에게 친숙한 소재에 의존한다. 이 적절 매우 큰 단지를 분리하는 능력에 제한되고, 보편적으로 성공하지 않은 반면, 방법은 잘 연구 단지의 다양한 캡처 할 수 있었고, 관심이 많은 시스템에 가능성이 적용됩니다.

이 데모 실험에서 다량 체-PAGE는 전체 쥐의 뇌 해물을 시행 하였다. 얻어진 단백질 분리 diflouride 폴리 비닐 (PVDF) 멤브레인에 블 롯팅 한 후, 복합체를 형성하는 것으로 알려진 단백질에 대한 항체로 프로빙 하였다. 도 1은 두 가지 방법에 의해 프로토콜의 유효성을 나타낸다. 먼저, 가교 결합 단백질 가교 시약 형성된 관찰 고 분자량 종을 의미 환원제의 첨가에 ...

Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

안정화 신규 이차원 폴리 아크릴 아미드 겔 전기 영동에 결합 된 아민 반응성 단백질 가교제를 사용하여 변성 조직 파쇄물로부터 천연 단백질 복합체를 분리하는 방법 (PAGE) 시스템이 제공된다.

다량 체-PAGE : 생물학적 샘플에서 캡처하는 방법 및 해결 단백질 단지

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

multimer-page-method-for-capturing-resolving-protein-complexes

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

11.3K 조회수.  Michigan State University.  안정화 신규 이차원 폴리 아크릴 아미드 겔 전기 영동에 결합 된 아민 반응성 단백질 가교제를 사용하여 변성 조직 파쇄물로부터 천연 단백질 복합체를 분리하는 방법 (PAGE) 시스템이 제공된다. 

비디오: Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled 포유 동물 세포에서 단백질 단지 선호도 캡처

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

연구

생화학

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 ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

동적 단백질 복합물 분석에 조립 하 고 질량 분석 및 전자 현미경을 사용 하 여 Biolayer 간섭계 바이오 센서에서 발표

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

BN-MS 분석에 의한 고분해능 컴플렉스 프로파일링

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

대규모 핵 상호 작용 프로파일링을 위한 라벨 없는 면역 침전 질량 분광 워크플로우

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

불안 정한 다중 단백질 단지의 분리에 의해 생체내에 제어 세포 간 연결 및 단백질 자기 동질 크로마 토그래피

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

생물학

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

구조 질량 분광법에 의해 큰 단백질 단지 분석

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

단백질 상호 작용을 발견하고 HaloTag 기술을 사용하여 단백질의 기능을 특성화

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...