Name: tandem affinity purification of protein complexes from eukaryotic cells
Uploaded: 2017-01-26T14:00:00.000+00:00
Duration: 11 min 30 s
Description: Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1. 도금 세포 <ol><li> 전날 형질에 10 % (v / v)의 소 태아 혈청 (FBS), 1 % (v / v)의 페니실린 / 스트렙토 마이신이 보충 된 둘 베코 변형 이글 중간 (DMEM) 10 mL에 3.5 × 106 HEK293T 세포 플레이트 100mm 접시 당 (10,000 U / ㎖의 주). 플레이트 (3) - 실험 / 상태 당 5 100mm 요리는 충분한 단백질 복구를 보장합니다. 주 : 판의 개수는 관심있는 단백질 복합체의 발현 수준과 용해도에 의존하는 각 실험 / 조건에 대해 결정되어야한다. 대규모의 정제가 필요한 경우에는 세포주 스피너 플라스크이 현탁액에서 성장하도록 적응 될 수 있지만,이 방법은 일시적 형질 감염을 위해 작동하지 않을 것이다. </li></ol> 2. 안정 세포주를 세포 형질 감염 또는 유도 <ol><li> 다음 날, 현미경으로 세포를 확인합니다. proce을하기 전에 80 % 합류 - 세포는 70해야합니다대한 수정 사항. </li><li> 100mm의 접시 당 형질 감염 시약 7 플라스미드 DNA의 μg의 21 μl의 총 혼합물로 세포를 형질 감염. 형질 감염 효율에 대한 제어는 벡터 표현 GFP (표 1)과 함께 형질 감염 세포. 주 : 독시사이클린 14, 15에 대응하여 두 개 이상의 복잡한 소단위의 발현을 유도 HEK293-T-REX 또는 유사한 안정한 세포주를 사용하는 경우, 유도제는 세포에 첨가하고 48 시간 동안 배양되어야 할 것이다. 마찬가지로, 유도제의 첨가에 따라 GFP의 발현을 유도하는 안정한 세포 라인은 또한 검증을 위해 요구된다. 2.7 단계로 진행합니다. </li><li> 100mm 접시 당 형질 전환 혼합물을 준비합니다. 1.5 ml의 마이크로 원심 튜브 (둘 이상의 플라스미드에 상당)의 총 플라스미드 DNA 7 μg의로 비추 DMEM 500 μL를 혼합한다. 별도의 microcentrifuge 관에서 비추 DMEM 위스콘신의 500 μl를 혼합트랜 시약의 제 21 μL. 각 형질 전환 반응에 대해 반복합니다. </li><li> 플라스미드 DNA 용액으로 형질 감염 시약 용액을 피펫 (역순으로 용액을 혼합하지 않음). 적어도 4 회 피펫 팅하여 혼합한다. </li><li> 10 시간 동안 실온에서 혼합물을 부화하지 - 15 분, 그러나 더 이상 15 분 이상. </li><li> 미디어 저장소를 생성하고 조심스럽게 세포를 방해하지 않고 판의 내부면에 드롭 현명 형질 전환 혼합물을 피펫 할 수있는 판을 기울입니다. 원래 평평한 위치에 접시를 반환하고, 혼합물을 배포하는 부드럽게 소용돌이 친다. </li><li> (이 조건은 이하 &quot;조직 배양 인큐베이터&quot;라 함), 5 % CO 2 습윤 세포 배양 인큐베이터에서 37 ℃에서 세포를 인큐베이션. 미디어 5 시간 후 형질 전환 (옵션) 장착합니다. </li></ol> 3. 검사의 형질 또는 유도 효율 <ol><li> 24 시간 후 형질에서, 독감에서 세포를 시각화orescent 현미경은 형질 전환 효율 (또는 GFP를 발현 제어 HEK293T 렉스 안정적인 세포주의 유도)를 확인합니다. 또 다른 24 시간 동안 품어. </li></ol> 4. STREP 선호도 정화 (STREP AP) <ol><li> 세포를 수집 <ol><li> 48 시간 포스트 형질에서 미디어를 제거하고 차가운 1X 인산 완충 식염수 (PBS)의 8 ml를 추가합니다. 접시에서 세포를 분리 아래로 피펫합니다. </li><li> 수집하고, 15 ㎖의 튜브에 세포 현탁액을 전송하고 4 ℃에서 4 분 동안 800 XG에서 세포를 스핀 다운. </li><li> PBS를 상층 액을 제거합니다. 잔여 PBS를 제거하기 (4 ℃에서 1 분 동안 800 XG)를 스핀을 반복합니다. 나중에 사용하기 위해 -80 ° C에서 세포 펠렛을 저장 또는 단백질 정제를 계속하려면 다음 절차를 따르십시오. </li></ol></li><li> 세포를 용균 <ol><li> 패시브 용해 완충액을 세포 펠렛 부피 × 5를 사용하여 세포 펠릿 (~ 플레이트 당 250 μL)에 재현 탁(PLB : 20 mM의 pH 7.5 인 Tris-HCl, 150 mM의 염화나트륨, 1.5 밀리미터의 MgCl 2, 1 mM의 DTT, 프로테아제 억제제 칵테일 정제, 5 % V / V 글리세롤, 1.0 % NP-40) 및에 세포 현탁액을 전송 1.5 ml의 microcentrifuge 관. </li><li> 간단히 4 ° C에서 30 분 동안 정지 및 nutate을 소용돌이. </li><li> 4 ° C에서 10 분 동안 10,000 XG에서 세포 현탁액을 스핀. </li><li> 새로운 1.5 ML의 microcentrifuge 관에 용해 용 해물을 전송하고 세포 펠렛을 폐기합니다. 같은 수용성 해물의 최종 부피의 10 % 할인 &quot;연쇄상 구균 AP 입력.&quot; 4 ℃에서 보관하십시오. </li></ol></li><li> 연쇄상 구균 구슬을 평형화 참고 : 첫 번째 AP 단계에 대한 사용 STREP 구슬! 연쇄상 구균 구슬 FLAG 구슬 아래로 끌어 것보다 훨씬 더 많은 단백질을 복구와 단지 아래로 당겼다. <ol><li> 꺼내 (n은 40 μl를 ×) + n은 50 % 연쇄상 구균 비드 슬러리의 μL (N = 샘플 수), 4 ℃에서 2 분 동안 1,500 XG에 스핀 다운. 참고 : 넓은 m를 사용하여피펫 구슬에 outhed 팁. </li><li> 상층 액을 제거하고 구슬에 PLB의 500 μl를 추가합니다. 4 ℃에서 5 분 동안 구슬 혼합물을 Nutate. 4 ° C에서 2 분 동안 1,500 XG에 스핀 다운. <ol><li> 3 세척 총 반복합니다. 40 μL × N의 최종 부피로 PLB의 비드를 재현 탁. </li></ol></li><li> 4 ° C에서 2 시간 동안 각각의 세포 용 해물 샘플 및 nutate 50 % 연쇄상 구균 비드 슬러리의 40 μl를 추가합니다. </li></ol></li><li> STREP AP <ol><li> 4 ℃에서 2 분 동안 1,500 XG에서 솔루션을 스핀 다운. </li><li> 4 ° C에서 &quot;STREP AP 플로우를 통해 (FT)&quot;로 뜨는 및 저장소를 제거합니다. </li><li> I (20 mM 트리스 - 염산의 pH 7.5, 250 mM의 염화나트륨, 1.5 밀리미터의 MgCl 2, 1 mM의 DTT, 5 % 글리세롤 및 0.2 % NP-40) STREP AP 세척 버퍼 구슬에 500 μl를 추가합니다. 4 ℃에서 2 분 동안 1,500 XG에서 5 4 ° C에서 분 원심 분리기의 구슬 혼합물을 Nutate. 상층 액을 버린다. <ol><li> 네 세척의 총 반복합니다. <br/&gt; 주 : 네번째 세척 전에 단백질 배경을 감소시키는 새로운 1.5 ML의 마이크로 원심 튜브에 비드 용액 옮긴다. 이 단계는 필수적이다. </li></ol></li><li> 500 μL 세척 버퍼 II (100 mM 트리스 - 염산의 pH 8.0, 150 mM의 염화나트륨, 1 mM의 EDTA)를 추가하고 튜브를 반전 구슬을 평형. 4 ° C에서 2 분 동안 1,500 XG에 스핀 다운 및 상층 액을 버린다. </li><li> STREP 용출 버퍼 (100 mM 트리스 - 염산의 pH 8.0, 150 mM의 염화나트륨, 1 mM의 EDTA, 2.5 mM의 Desthiobiotin)의 50 μL과 관심의 단백질을 용출. 4 ℃에서 15 분 동안 800 rpm에서 소용돌이. </li><li> 4 ° C에서 1 분 동안 1,500 XG에서 구슬을 스핀 다운. 상층 액을 수집합니다. 이 부분은 &quot;STREP AP 용출&quot;샘플에 해당합니다. </li><li> 제 1 용출 얻어진 단백질의 양의 절반까지 얻을 수있는 초 용출, 4 ° C에서의 비드 부분을 저장한다. 실버 염색 및 / 또는 웨스턴 블로 팅에 대한 STREP AP 용출의 나누어지는 10 %16. </li></ol></li><li> 국기 구슬을 평형화 <ol><li> (NX 16 μL) + n은 FLAG 비즈 솔루션의 μL (샘플 N = 번호)를 꺼내 4 ℃에서 2 분 동안 1,500 XG에 스핀 다운. 참고 : 피펫 구슬에 입이 큰 팁을 사용합니다. </li><li> 상층 액을 제거하고 FLAG 세척 버퍼 500 μL를 추가 (100 mM 트리스 - 염산의 pH 8.0, 150 mM의 염화나트륨, 1 mM의 EDTA, 0.1 % NP-40) 구슬에. 4 ° C에서 2 분 동안 1,500 XG에 스핀 다운. 3 세척 총 반복합니다. </li><li> NX 16 μL의 최종 부피 FLAG 세척 완충액으로 비드를 재현 탁. </li><li> 4 ° C에서 나머지 STREP AP 용출 및 nutate 하룻밤에 FLAG 비드 슬러리의 16 μl를 추가합니다. </li></ol></li></ol> 5. FLAG IP <ol><li> 4 ° C에서 2 분 동안 1,500 XG에서 플래그 구슬 서스펜션을 스핀. 4 ° C에서 &quot;FLAG IP 플로우를 통해&quot;(FT)으로 뜨는 및 저장소를 저장합니다. </li><li> 500 추가(81); 솔루션에 FLAG 세척 버퍼의 리터. 4 ° C에서 2 분 동안 1,500 XG에 5 4 ° C에서 분 원심 분리기에 대한 Nutate. 네 세척의 총 반복하고 각 세척 한 후 상층 액을 제거합니다. 주 : 네번째 세척 전에 배경을 감소시키는 새로운 1.5 ㎖의 마이크로 원심 튜브에 단백질로 비드 용액 옮긴다. 이 단계는 필수적이다. </li><li> 마지막 스핀에서 뜨는을 제거하고 구슬로 FLAG 펩타이드의 200 NG / μl를 포함 FLAG 세척 버퍼의 30 μL를 추가합니다. 4 ℃에서 2 시간 동안 800 rpm에서 소용돌이. </li><li> 4 ° C에서 2 분 동안 1,500 XG에서 정지를 스핀 다운. 으로 4 ° C에서 뜨는 및 저장소를 수확 &quot;FLAG IP 용출.&quot; 참고 : 용출 샘플 표준 프로토콜 16을 사용하여 실버 얼룩 및 / 또는 웨스턴 블롯으로 확인하고, 또한 단백질 분석을위한 준비가 될 수있다. (1) STREP AP 입력, (2) STREP AP FT, (3) STREP AP Eluti : 웨스턴 블롯을 설정할 때, 다음의 모든 샘플을 실행(4) FLAG IP FT, (5) FLAG IP 용출에. 로드 볼륨은 각 실험에 대해 결정되어야 할 것이다. 수집 된 모든 샘플과 비드 단기 기억과 장기 보존 -80 ° C에서 39 ° C에서 저장 될 수있다. TAP 샘플들은, 그 구성 요소의 신원을 확인하는 신규 번역 후 변형 (PTMS)를 식별 및 / 또는 정제 착체 2 (17)와 임의의 신규 단백질 상호 작용을 발견 질량 분석법에 의해 분석 될 수있다. 이러한 목적하는 쿠마 겔 또는은 염색을 실행 한 후, 밴드 깨끗한 메스를 사용하여 겔로부터 절제 될 수있다. 질량 분석 방법을 논의 몇 가지 우수한 리뷰 이전에, 19 (18)을 발표했다. </li></ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KAP1+Recruitment+of+the+7SK+snRNP+Complex+to+Promoters+Enables+Transcription+Elongation+by+RNA+Polymerase+II.">KAP1 Recruitment of the 7SK snRNP Complex to Promoters Enables Transcription Elongation by RNA Polymerase II.</a> Mol Cell. 61, 39-53 (2016).</li><li>Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (1994).</li><li>Trudgian, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+label-free+SINQ+normalized+spectral+index+quantitation+in+the+central+proteomics+facilities+pipeline.">Comparative evaluation of label-free SINQ normalized spectral index quantitation in the central proteomics facilities pipeline.</a> Proteomics. 11, 2790-2797 (2011).</li><li>Choudhary, C., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+signalling+networks+by+mass+spectrometry-based+proteomics.">Decoding signalling networks by mass spectrometry-based proteomics.</a> Nat Rev Mol Cell Biol. 11, 427-439 (2010).</li><li>Bensimon, A., Heck, A. J., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+proteomics+and+network+biology.">Mass spectrometry-based proteomics and network biology.</a> Annu Rev Biochem. 81, 379-405 (2012).</li><li>Rogers, S., Wells, R., Rechsteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+sequences+common+to+rapidly+degraded+proteins:+the+PEST+hypothesis.">Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.</a> Science. 234, 364-368 (1986).</li></ol>

The authors have nothing to disclose.

발현 및 진핵 세포에서 단백질 복합체의 분리를 위해 여기에 설명 된 프로토콜은 분자 어셈블리의 생화학 적 특성에 한정되지 않고, 그 기능을 조절할 수있는 신규 인터랙 번역 후 변형의 식별에 이용 될 수있다. 친 화성 태그의 활용은이 프로토콜에서 언급 한 내용에 제한되지 않는다; 그러나 우리의 경험은 제 AP 단계로 STREP을 사용하여 상당히 최종 단백질 - 단백질 복합체의 수율을 향상하는 것...

<html> 단백질 - 단백질 상호 작용 (프로톤 펌프 억제제)는 그 기능이에 대해 알릴 수 생물학적 과정 (1),이 프로톤 펌프 억제제에 대한 추가 연구의 정확한 조절을 위해 중요하다. 몇몇 접근법은 프로톤 펌프 억제제의 연구 및 특성뿐만 아니라 신규 조절 단백질 성분의 드 노보 식별을 위해 고안되었다. 1989 년 스탠리 필드와 동료들은 효모 두 하이브리드 (Y2H) 분석 (3) 보도했다. 이 방법은 사카로 마이 세스 세레 비지에 인터랙 관심 (미끼)의 정의 단백질 (할까요)의 공평하고 포괄적 인 식별 할 수 있습니다. 프로톤 펌프 억제제를 발견하기위한 현저한 유틸리티 또한 Y2H 분석은, 효모 세포에서 단백질을 특성화 쌍 최소 상호 작용 도메인을 정의하고, 이러한 상호 작용을 폐지 변이를 식별하는데 사용될 수있다. Y2H 분석을 수정하여, 프로톤 펌프 억제제는 포유 동물 세포에서 공부하실 수 있습니다클래스 = &quot;외부 참조&quot;&gt; 4. Y2H 분석 (예, 효모 세 하이브리드 시스템)의 변형, 단백질 및 RNA 세포 단백질 작은 유기 리간드 상호 작용을 연구에 적용 할 수있다. 상동 시스템에서 프로톤 펌프 억제제를 연구하는 또 다른 일반적으로 사용되는 도구는 공동 면역 (공동 IP) 분석 5입니다. 관심의 단백질을 면역 침전에 대한 항체를 사용하여, 공동 IP 분석 연구자들은 다양한 환경 조건 및 실험 상황에서 셀의 프로톤 펌프 억제제를 모니터링 할 수있다. 에피토프 - 태그 단백질 (예를 들면, FLAG는, Myc와, STREP 및 HA는, 다른 사람의 사이에서) 친 화성 정화 (AP) 방법이 서양 얼룩 등 여러 다운 스트림 분석 복잡한 단백질 혼합물에서 단백질의 분리를 촉진했다,은 얼룩의 사용 및 효소 분석. 그러나 이러한 이전 방법 중 어느 것도 체외을 포함하여 더 특성화 단백질 복합체의 대량의 분리를 사용하지ssays, 질량 분석 및 번역 후 변형의 식별에 의한 규제 서브 유닛의 발견. AP 통신 방식의 향상된 버전은 두 개의 연속적인 AP들 (6, 7)를 통해 정제하여 두 에피토프와 융합 단백질을 생성하여 프로톤 펌프 억제제의 연구를위한 정제 기술이다 텐덤 AP (TAP)를 호출한다. 이 문서에서 우리는 두 개의 서브 유닛이 다른 에피토프 태그 다음 두 순차 액세스 포인트 (AP STREP FLAG IP 다음)를 통해 정제하는 정제 단백질 복합체의 TAP 방법의 변화를 제시한다. 연구자들이 관심을 자신의 단백질 복합체에 적용 할 수 있도록 우리가 먼저, TAP의 최소한의 개요 (그림 1)과 모든 실험 단계에 대한 자세한 설명 (그림 2)을 제공한다. 탭 방식의 적용 성을 입증하기 위해, 우리는 (복소 잘 특징 사이클린 CDK는 PT라고 선택한규제 소단위 사이클린 T1 (CycT1) 및 키나제 (경우, Cdk9)로 구성되며, RNA 중합 효소 II (POL II) 8, 9, 10에 의해 전사 조절에 관여 EFB 키나아제). P-TEFb는 폴 II 상기 프로모터에서 전사를 일시 완화 연관된 부정적인 신장 요소의 C 말단 도메인을 인산화시켜 전사 신도 11, 12, 13을 용이하게한다. 이를 염두에 알려진 상호 작용, CycT1을 STREP - 태그 및 FLAG-태그 경우, Cdk9는 HEK293T 세포에서 발현 이상이었다. 상호 TAP 실험은 더 단백질 상호 작용이 사용 된 에피토프에 독립적 인 것을 확인하는 STREP-태그 경우, Cdk9과 FLAG-태그 CycT1로 하였다. 세포를 수거하고, 48 시간 후 형질 감염을 용해시켰다. 가용성 해물이 TAP 그래피 (STREP AP는 FLAG 다음IP). 입력 및 정제 된 단백질은 서양 얼룩과 실버 얼룩 (그림 3)에 의해 분석 하였다.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH3002.2FS</td><td></td></tr><tr><td>Fetal bovine serum</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH30071</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>MP091670049</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen Laboratories</td><td>SL100688</td><td></td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11836153001</td><td></td></tr><tr><td>STREP-Tactin Superflow</td><td>IBA Lifesciences</td><td>2-1208-010</td><td></td></tr><tr><td>STREP-tag elution buffer</td><td>IBA Lifesciences</td><td>2-1000-025</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>F2426</td><td></td></tr><tr><td>Corning 100 mm &amp;times; 20 mm style dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td><td>Corning&amp;nbsp;</td><td>430167</td><td></td></tr><tr><td>Protein Lo-Bind Eppendorf</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-370</td><td></td></tr><tr><td>48 hole micro tube foam rack</td><td>Fisher Scientific</td><td>02-215-386</td><td></td></tr><tr><td>Labquake shaker rotisserie&amp;nbsp;</td><td>Thermo&amp;nbsp;</td><td>415110</td><td></td></tr></tbody></table>

tandem affinity purification of protein complexes from eukaryotic cells

활성 단백질 - 단백질, 단백질 - 핵산 복합체의 정제 효소 활성 및 신규 서브 유닛 및 번역 후 변형의 드 노보 식별의 특성에 매우 중요하다. 박테리아 시스템들은 단일 폴리펩티드 및 단백질 복합체의 다양한 발현 및 정제를 허용한다. 그러나,이 시스템은 번역 후 변형 (예를 들어, 아세틸 화 및 포스 포 릴화)을 포함 소단위 단백질의 정제 및 진핵 시스템에서의 발현 만 존재하는 신규 규제 소단위 /의 식별을 가능하게하지 않는다. 여기서 우리는 진핵 세포에서 일시적으로 또는 안정적으로 표현 에피토프 - 태그 단백질과 단백질 복합체의 정제를 용이하게하는 소설, 강력한, 그리고 STREP-를 사용하여 효율적인 탠덤 선호도 정화 (TAP) 방식과 FLAG-태그 단백질에 대한 자세한 설명을 제공합니다. 이 프로토콜은 PROT의 특성을 적용 할 수있다EIN 복잡한 기능 복잡한 서브 유닛의 번역 후 변형을 발견, 및 질량 분광법에 의해 신규 규제 복잡한 구성 요소를 식별하기. 특히,이 방법은 TAP 따라서 하류 실험 기회 광범위한 항복 진핵 또는 병원성 (바이러스 성 및 세균성) 구성 요소에 의해 형성되는 단백질 복합체를 연구에 적용될 수있다. 우리는 단백질 복합체와 협력 연구자들이 다양한 방법으로이 방법을 활용할 수 있다고 제안한다.

이 글에서, 우리는 (또한 P-TEFb 키나제라고도 함) 복잡한 잘 특징 CycT1-경우, Cdk9에 TAP 방법의 적용 가능성을 보여줍니다. 그리고 경우, Cdk9-FLAG (경우, Cdk9 : F), 또는 경우, Cdk9-STREP (경우, Cdk9 : S) 및 시클 T1-FLAG (CycT1 : F) : 시클 T1-STREP (S CycT1) 인코딩하는 플라스미드 (표 1), HEK293T 세포로 형질 감염시켰다 . F 또는 CycT1 : <s...

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Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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 ...

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Biochemistry

14.9K 조회수.  The University of Texas Southwestern Medical Center. 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.

비디오: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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 ...

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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 ...

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 ...

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 ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

탠덤 동질 정제를 사용하여 단백질 나누기 파트너의 식별

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

생물학

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 ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

단백질 - 단백질 상호 작용을 확인<em&gt; 초파리</em탠덤 선호도 정화로&gt; 성인 머리 (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

탠덤 선호도 태그 방법을 사용하여 구조 연구에 대한 기본 공단의 정화

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

단백질-단백질 상호작용을 연구하기 위한 Tandem Affinity Purification Assay

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

진핵 세포에서 단백질 복합체의 탠덤 선호도 정화

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