This work was funded by the Wellcome Trust (WT098051).

<ol>
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The authors have nothing to disclose.

여기, 우리는 단백질 복합체를 해결하기 위해 양적 질량 분석과 함께 파란색 기본 겔 전기 영동 다음에 친화력 정화의 사용을 설명합니다. 이 방법은 기능성 단백질 어셈블리로 일차원 단백질 상호 작용의 목록을 해명하기위한 방법을 제공한다. 우리는 에피토프 - 태그 단백질의 사용을 기반으로하는 방법을 보여줍니다. 태그가 단백질을 발현하는 세포주를 사용할 수없는 경우, 대안은 관심의 단백질에 대한 항체를 사용하는 네이티브 경쟁 용출을 달성 할 수 펩타이드가 제공 할 수도 있습니다. 출발 물질과 구슬의 양의 크기는 목적 단백질의 발현 수준에 따라 수정해야 할 수도 있습니다. 우리는 일반적으로 출발 물질 2-5 × 108 셀이 프로토콜을 수행하고, 이는 심지어 낮은 발현 수준을 갖는 단백질을 충분하다. 용해 완충액의 조성은 달성하도록 실험적으로 선택되어야근처 미끼의 가용화를 완료합니다. 이 바인딩 특정 염색질 또는 막 단백질에서 단백질의 일부 ​​유형에 대한 도전이 될 수 있습니다. 대안적인 옵션은 연구중인 단백질 복합체는 높은 염 또는 염색질 결합 단백질 20, 29, 초음파 및 / 또는 뉴 클레아 제 처리를 사용하여 안정한 것이어야한다 소금의 양을 증가시키는 것을 포함한다. 막 단백질의 경우, DDM 또는 디기 토닌에 세제를 전환하는 것이 바람직 할 수도있다 (30). 이 DNA 결합 단백질의 경우, 정제 단계 (20) 중 벤조 나제 바와 같은 뉴 클레아 제를 포함하는 것이 유용하다. 핵산의 완전한 제거는 검출 된 상호 작용 단백질과 DNA 사이에서 발생에 의해 매개되지 않는 것을 보장한다. 이 방법을 사용하여 조사중인 착체의 안정성이 때 중요한 요소를 고려한다. 절차는 길고 preservin을 포함 할 수있다g 단백질 복합체 밤새. 우리는이 크로 마틴 리모델링 복합체 (D 보데와 M 파르도, 데이터가 표시되지 않음) 좋은 성공을 달성,하지만이 평가되어야한다. 필요한 경우 친화 정제 공정을 단축 할 수있다. 같은 크기 배제 크로마토 그래피 등의 기본 분류를 허용 대체 기술은 널리 단백질 복합체의 특성을 50 년 이상 사용되어왔다. 우리는 청색 등 천연 PAGE의 해상도가 크기 배제 크로마토 그래피 20, 31, 32, 33을 사용하여 달성이 우수하다는 것을 보여 주었다. 블루 기본 페이지의 또 다른 장점은 비용이 크로마토 그래피 시스템을 필요로하지 않는, 오히려 실험실에서 널리 퍼져 단백질 전기 영동 장비를 사용합니다. 의 측면에서 손에 시간,이 방법은 기존의 geLC-MS / MS (A)보다 더 많은 작업을 포함하지 않습니다pproach 또는 오프라인 크로마토 그래피 분별. 대부분의 분류 기술 할 그러나, 그것은 그들의 해상도에 한계가 있습니다. 매우 균일 또는 질량과 모양에 가까운 단지는 파란색 기본 페이지에서 제공하는 해상도 및 공유 서브 유닛과 별개의 단지를 해결 보편적으로 성공하지 않을 수 있습니다 여기에보고 따라서 프로토콜을 넘어 수 있습니다. 격려하여, 우리는 세 개의 서브 유닛 (M 파르도, 준비 원고를) 공유하는 두 개의 매우 유사 사량 단지를 분리하는데 성공했다. 질량 분석 점점 민감한되어 있기 때문에, 불특정 인터랙 오염물 미세한 양 AP-MS 샘플에서 검출 될 수있다. 여기에 제시된 방법은보다 높은 분자량에서 미끼 마이그레이션 피크와 일치 마이그레이션 봉우리 단백질에 관심을 집중하여 친화력 정화의 배경 오염 실제 인터랙의 차별에 도움이 있습니다단량체 단백질. 몇몇 그룹이 이전의 분리 10, 11, 12, 34에 대한 필요없이 셀룰러 규모 단백질 복합체를 묘사하는 단백질 상관 프로파일이어서 분별 기법을 사용 하였다. 그러나,이 하위 화학 양 론적 상호 작용을 검출하는 오류가 발생할 수 있습니다. 친화력 정화를 통해 농축 단계의 통합이를 극복하는 데 도움이 될 수 있습니다. 여기에 설명 된 방법은 특정 단백질이 동일한 셀룰러 컨텍스트 내의 소요 부분에 상기 다수의 복합체 단백질 복합체의 토폴로지 탐색 및 해명에 일반적으로 유용한 것으로한다. 이 전략은 단백질 복합체를 해결하기 위해 고가의 크로마토 그래피 분별 장비가 없을 수 있습니다 실험실 간단하고 의무입니다.

세포에서, 가장 단백질은 일시적인 단백질 - 단백질 상호 작용을 통해 안정한 단백질 또는 조립체를 형성하여 기능을 수행한다. 단백질 상호 작용을 특성화하는 것은 충분히 이해 세포 과정에 매우 중요합니다. 질량 분석 (AP-MS)와 함께 선호도 정화는 천연 단백질 상호 작용을 식별하기 위해 가장 일반적으로 적용되는 전략 중 하나입니다. 지난 10 년간 달성 기기의 기능을 크게 향상이 방법은 매우 강력한 만들었습니다. AP-MS 실험에 의해 확인 된 상호 작용이 미끼와할까요 사이의 직접 및 간접 협회의 혼합물을 포함하는 것이 중요합니다. 또한, 종종 단백질은 다른 생물학적 역할을 할 수도 있습니다 동일한 세포 맥락에서 여러 가지 다른 단지에 참여, 따라서 AP-MS에 의해 식별됩니다 인터랙는 별개의 단백질 어셈블리 또는 기능 엔티티의 혼합을 나타낼 수 있습니다. 도출 할 수 없습니다이러한 토폴로지 정보는 AP-MS 간단한 실험에 의해 생성 된 단백질의 모노 차원 목록에서 선험적. 그러나,이 기술은 이러한 어셈블리를 해결하기 위해 하나 이상의 방법과 결합하여 단백질 복합체의 구조를 정의하는 데 더 이용 될 수있다. AP-MS를 통해 확인 된 단백질 상호 작용의 토폴로지를 해결하기 위해 몇 가지 전략이 적용되고있다. 하나의 방법은 실험 1과 베이트 이전 라운드에서 확인할까요을 이용하여 반복적 인 AP-MS 실험을 수행한다. 매우 유익한 있지만,이 두 실험 및 해석 상당히 노동 집약적 인 작업입니다. 질량 분석기와 결합 단백질 가교 점점 단백질 복합체 2, 3, 4, 5에 대한 위상 정보를 도출하는 데 사용되고있다. 그러나 computatio가교 결합 된 펩티드의 최종 분석은 여전히 ​​도전 과제로 남아 따라서 흐름의 병목 현상이다. MS 분해성 가교 시약의 출현 단백질 (6, 7)를 상호 작용에 가까운 위치에 아미노산 잔기의 매핑을 용이하게한다. 또 다른 방법은 종래의 직교 분리 기술 8,9 친화 정제를 조합하는 것이다. 겔 여과 나 이온 교환 수, 또는 수 크로스 구배 분획 법에 의해 크로마토 그래피 분별은 또한 최근에 의해 통과 복잡한 분리 단계 10, 11, 12, 13, 시스템 전체의 레벨에서 multiprotein 착물을 설명하기 위해 정량적 질량 분석과 조합하여 사용되고있다. 블루 네이티브 폴리 아크릴 아미드 겔 전기 영동 (BN-PAGE)는 널리 적용되었습니다천연 단백질 상호 작용, 미토콘드리아 막 단백질 복합체 (14)를 포함하는 일반적으로 사람들을 조사합니다. 이 분리 기술은 최근 미토콘드리아 단지 15 일에뿐만 아니라 라벨이없는 단백질 정량과 상관 프로파일 링과 함께 사용 된 16, 17,뿐만 아니라 19 일 전체 세포 18에서 다른 단백질 복합체를 푸는합니다. 우리는 이후 기본 분류 방법과 정량적 인 MS와 친화력 정화의 조합이 특정 단백질을 포함하는 여러 단백질 어셈블리를 해결하기위한 유용한 전략을 제공해야한다는 가설을 세웠다. 여기서 우리는 정량적 질량 SP이어서 절연 단지 청색 기본 폴리 아크릴 아마이드 겔 전기 영동법으로 일반 에피토프 계 친화 정제를 조합하는 방법을 설명ectrometry 단백질 상관 프로파일은 단백질에 관여 할 수있는 여러 어셈블리를 해결합니다. 우리는 에피토프 태그에 융합 관심의 단백질이 생리적 풍부 가까이 달성하고 효율적인 기본을 보장하기 위해 내생 로커스에서 표현 마우스 배아 줄기 세포를 사용 복잡한 격리. 이 방법은 단백질이 기존의 geLC-MS (20)보다 더 많은 작업을 필요로하지 않는 동안, 자신의 상관 프로필을 기반으로 별개의 어셈블리에 이러한 문제를 해결하기에 종사 여러 상호 작용을 풀어.

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Protein G Dynabeads</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">10003D</td>
			<td style="width:452px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">FLAG antibody M2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Tween-20, protein grade, 10% solution</td>
			<td style="width:285px;">Millipore</td>
			<td style="width:220px;">655206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Dimethyl pimelimidate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">80490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">0.2 M Triethanolamine buffer, pH 8.2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">T0449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">3x FLAG peptide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Vivaspin 5K NMWCO, PES</td>
			<td style="width:285px;">Sartorius</td>
			<td style="width:220px;">VS0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativePAGE 3-12% Bis-Tris gel</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Running Buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Cathode Additive</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">4x NativePAGE sample loading buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativeMARK</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">LC0725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NativePAGE 5% G-250 sample additive&#160;</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc conical bottom 96-well plate, polypropylene</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">249944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Multiscreen HTS 96-well filtration system</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">MSDVN6550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc 96-well cap natural</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">276002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Iodoacetamide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">I1149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Acetonitrile, HPLC grade</td>
			<td style="width:285px;">Fisher Scientific</td>
			<td style="width:220px;">A/0627/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Trypsin, sequencing grade</td>
			<td style="width:285px;">Roche</td>
			<td style="width:220px;">11418475001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Software</td>
			<td style="width:285px;"></td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MaxQuant (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111795/maxquant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Perseus (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111810/perseus</td>
		</tr>
	</tbody>
</table>

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.

질량 분석에 의해 친 화성 정제 블루 천연 단백질 상관 프로파일의 워크 플로우 (ABC-MS) 전략은도 7에 도시되어있다. 관심의 단백질 주변의 원주민 단백질 복합체 (이 경우 FLAG)에 에피토프 태그에 대한 항체를 사용하여 친화력 정화 및 경쟁력 용출에 의해 격리됩니다. 착물은 BN-PAGE에 의해 해결되며, 전체 겔 레인은 48 개 부분으로 절개하고, 샷건 LC-MS / MS를 위해 준비했다. MS 정량 정보는 청색 기본 분리 걸쳐 각각의 식별 된 단백질 마이그레이션 프로파일을 생성하는 데 사용된다. 상호 작용하는 단백질을 겹쳐 피크 구별 복잡한 디스플레이 유사한 이동 프로파일을 형성한다. 목적 단백질은 하나 개 이상의 부 조립에 걸리는 경우, 다수의 피크가, 그 이동 프로파일에서 관찰 된 서브 착물 청색 네이티브 겔 해상력 내에 주어진다. 미그의 체계적인 비교배급량 프로필 계층 클러스터링을 사용하여 단백질의 프로파일 상관에 의해 달성 될 수있다. dendrogram은 모든 분획에 대한 피크 강도가 별개의 단백질 복합체에 속하는 단백질 (도 8)의 상호 작용의 확인을 용이하게 열지도 시각화된다. 우리는 MTA2, 복잡한 20 개조 NuRD 염색질의 핵심 서브 유닛의 상호 작용 파트너를 분석하기 위해이 전략을 사용했다. 도 9에 도시 된 바와 같이, MTA2의 이동 프로파일은 더 풍부 표시 하부 질량 피크, 700 kDa 이상 1.2 MDA 간의 구별 강도의 두 피크를 표시. MTA1 / 3 HDAC1 / 2 Mbd3 포함한 기타 NuRD 코어 서브 유닛은, 상기 서브 유닛, 즉 Chd4의 일부 피크이라도 동일한 분리 패턴을 보였다 B Gatad2a / 및 Rbbp4 / 7은 (도 9a 역 풍부 분포 있었다 B). 한편, CDK의 프로필2ap1,이 Wnt 유전자 프로모터 (23)에 NuRD 복합체를 모집 규제 인자 만 높은 질량 피크 (도 9c)를 표시하고 있으므로 Sall4도 NuRD 20, 24, 25에 결합하는 것으로 나타났다 전사 리프레했다. 따라서, 청색 기본 PAGE에 의한 친 화성 정제 MTA2 관련 단백질 분획은 NuRD 복합체의 두 가지 형태를 확인할 수 있었다. ABC-MS는 특정한 단백질 엔티티에 할당하면서 신규 인터랙의 식별을 허용한다. 열 -지도에 표시된 단백질 클러스터 및 분획 강도의 시험을 통하여 우리는 두 NuRD 피와 일치 개의 이전 피크를 표시 NuRD 소단위와 Wdr5와 Wdr5 상기 MLL의 메틸화 착체 (26)의 조절 서브 유닛 사이의 강한 상관을 검출Wdr5 및 NuRD 간의 새로운 상호 작용을 제안 eaks (도 8). 우리는 크기 배제 크로마토 그래피 (20)의 공동 면역와 공동으로 마이그레이션하여이 상호 작용을 확인했다. 계층 적 클러스터링 디스턴스 메트릭 계산의 선택은 클러스터 따라서 상관의 형태에 영향을 미칠 것이다. 우리는 단백질 또는 관심의 복잡한의 기존 상호 작용의 지식을 가진 가장 적합한을 달성하기 위해 서로 다른 측정 실험을하는 것이 좋습니다. 일반적으로 우리는 맨하탄 (L1)의 거리 메트릭 (20)에 최적의 결과를 달성했다. 피어슨 상관 또는 유클리드 거리 메트릭은 다른 분류 기법 10, 11, 12에 기초하여 복합체를 식별하기 위해보고되었다. 관음청색 기본 분획으로부터 수득 titative MS 데이터는 단백질 복합체의 화학량 론을 결정하는 데 사용될 수있다. iBAQ (강도 계 절대 정량) 값은 식별 된 단백질 (27), (28)의 상대적인 풍부함의 측정치를 제공한다. 마이그레이션 프로파일 내의 모든 피크 분획에서의 단백질의 복합 부재 iBAQ 값은 상대적인 양을 얻기 베이트 단백질의 정규화된다. 주어진 단백질 복합체 부재의 프로파일 피크 걸쳐 정규화 iBAQs 수평 흐름을 따라야하고, 그래프 값 미끼 단백질에 대해 상호 작용하는 단백질의 화학량 론을 반영한다. 화학량 론 계산의 더 상세한 표현, 20 참조. <img alt="그림 7" src="/files/ftp_upload/55498/55498fig7.jpg" /> 도 7 : 도식 플로 요약 ABC-MS의trategy. 이는도 20에서 수정된다. <a href="http://ecsource.jove.com/files/ftp_upload/55498/55498fig7large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 8" src="/files/ftp_upload/55498/55498fig8.jpg" /> MTA2의 인터랙의 BN-PAGE 이전 프로파일도 8 계층 클러스터링. MTA2 관련 단백질은 자신의 마이그레이션 프로필의 유사성에 따라 클러스터했다. 오직 NuRD 착체를 함유하는 가열 - 맵의 서브 세트 (파란색 상자 묶인) 도시되어있다. 노란색 상자가 NuRD 단지와 Wdr5의 강한 상관 관계를 강조한다. 주석 분자량이 같은 젤에서 실행 단백질 표준의 이동 거리에서 추정되었다. 이 연구는 원래 생화학 및 모 20 미국 사회 분자 세포 프로테오믹스에 발표 된<html> lecular 생물학. <a href="http://ecsource.jove.com/files/ftp_upload/55498/55498fig8large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 9" src="/files/ftp_upload/55498/55498fig9.jpg" /> NuRD 소단위와 관련된 단백질도 9 BN-PAGE 이전 단면도. A) 동일한 강도의 패턴으로 존재하고 MTA2 NuRD 소단위. B) 상기 역 강도 패턴 MTA2 및 NuRD 소단위. 오직 고 분자량 NuRD 엔티티에 존재하는 C) 및 MTA2 Cdk2ap1. 주석 분자량은 단백질 표준의 마이그레이션 프로필에서 추정되었다. 이 연구는 원래 분자 세포 프로테오믹스 (20) 생화학 및 분자 생물학에 대한 미국 사회에 출판되었다.&quot;_blank&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="보충 파일" src="/files/ftp_upload/55498/proteinGroupsDB04.jpg" /> 보충 자료 : 샘플 데이터 세트. ABC-MS 실험에서 단백질 동정 및 정량 값을 포함 proteingroups.txt 파일 MaxQuant 유래. 이 연구는 원래 분자 세포 프로테오믹스 (20) 생화학 및 분자 생물학에 대한 미국 사회에 출판되었다. <a href="https://www.jove.com/files/ftp_upload/55498/proteinGroupsDB04.txt" target="_blank">이 파일을 다운로드하려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling at JoVE.com

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

여기에서 우리는 라벨 무료 양적 질량 분석을 사용하여 단백질 상관 프로파일 다음에 친 화성이 단백질 복합체의 정화 및 파란색 기본 페이지에서 자신의 분리를위한 프로토콜을 제시한다. 이 방법은 서로 다른 단백질 복합체에 상호 작용 체를 해결하는 데 유용합니다.

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 ...

resolving-affinity-purified-protein-complexes-blue-native-page

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Biochemistry

13.7K 조회수.  Wellcome Trust Sanger Institute.  여기에서 우리는 라벨 무료 양적 질량 분석을 사용하여 단백질 상관 프로파일 다음에 친 화성이 단백질 복합체의 정화 및 파란색 기본 페이지에서 자신의 분리를위한 프로토콜을 제시한다. 이 방법은 서로 다른 단백질 복합체에 상호 작용 체를 해결하는 데 유용합니다. 

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

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

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

진핵 세포에서 단백질 복합체의 탠덤 선호도 정화

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

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

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

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 ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

단백질 -RNA 복합체의 향상된 검출을위한 수평 겔 전기 영동

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

그들의 네이티브 셀룰러 환경에서 상황에 맞는 단백질 복합물의 분할-BioID-Proteomic 분석

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 1: 원스텝 단백질 단백질과 단백질 대사 산물 단지의 병렬 분석에 대 한 친 화력 정화

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

블루 기본 젤 전기 이동 법에 의해 Thylakoid 막 단백질 복합물의 분석

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

탭 태그를 사용 하 여 엽록체 Translocon 단백질 복합물의 친 화력 정화

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

경쟁 적정 형광 현미경 검사 법을 사용 하 여 녹음 방송 요인 DNA 바인딩 선호도의 높은 감도 측정

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

단백질 폴딩, 전송, 및 방사성 펄스 체이스 하 여 살아있는 세포의 저하의 분석

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...