우리는 우리에게 효모 TAP 발현 플라스미드를 보내는 EUROSARF 감사합니다. 우리는 또한 라이언 Labadens에서 편집의 도움에 감사합니다. 이 작품은 CW에 NIH / NINDS 부여 (R01NS070962)에 의해 지원되었다

1. UAS-TAP-태그 형질 전환 파리를 생성 <ol><li> pUAST-TAP-태그 DNA 구조를 생성합니다. <ol><li> (N-또는 C-말단) 미끼 단백질의 TAP 태그는 단백질의 구조 / 기능을 기반으로 융​​합되어야하는 가로 결정한다. 자세한 내용은 설명을 참조하십시오. </li><li> 각각 N-또는 C-말단 태그가 UAS-TAP의 도입 유전자를 생성하는 pUAST-NTAP 또는 pUAST-CTAP 벡터의 여러 복제 사이트 (MCS)로 관심의 유전자의 cDNA를 코딩 영역을 서브 클론. 상세한지도와 가능한 제한 사이트 및 독서 프레임의 그림 1을 참조하십시오. </li></ol></li><li> UAS-TAP-태그 형질 전환 초파리를 생성합니다. <ol><li> P 요소 매개 삽입 (11)를 이용하여 표준 프로토콜을 다음과 형질 전환 초파리를 생성합니다. 사출 서비스의 개수는 시판되고있다. </li><li> 각각의 형질 전환 라인에 신경 GAL4 드라이버 (즉 BG380-GAL4)를 건너 단백질 발현 수준을 결정서부 (퍼 옥시 다제 안티 퍼 옥시 다제 항체) 얼룩 및 / 또는 (안티 - TAP 항체) 면역 염색하여 각 라인의. 일반적으로, 내인성 단백질에 가까운 단백질 발현 수준이 유전자 변형 라인은 TAP 절차에 권장됩니다. 자세한 내용은 설명을 참조하십시오. </li><li> 그 유전자의 기능 상실 돌연변이 체를 사용할 수있는 경우 TAP-태그 도입 유전자의 기능을 확인하는 구조 실험을 PerformGAL4/UAS-based. 실질적으로 다음 TAP 실험의 돌연변이 표현형을 구출 할 수있는 유전자를 선택합니다. </li></ol></li></ol> 2. TAP 절차에 대한 샘플을 준비 <ol><li> 신경 GAL4 드라이버 (예 : BG380-인 Gal4) 및 비행 샘플의 확장을 용이하게하기 위해 선택 TAP-태그 유전자를 모두 전달하는 비행 주식을 생성합니다. GAL4 드라이버와 위의 조합이 생존과 성장 단점이 발생 드문 경우 UAS - 유전자 십자가의 F1의 자손을 수집합니다. &lt;/ 리&gt; <li> 작은 규모의 샘플을 수집하고 TAP - 태그 단백질을 가용화 용해 상태를 최적화 할 수 있습니다. <ol><li> 비이 온성 세제 NP-40 (0.1-1 %), NaDOC (0.1-1 %) 및 ​​트리톤 X-100 (0.05-0.5 %)의 조합을 사용하여 용균 버퍼들의 시리즈를 만든다. 자세한 내용은 표 1과 설명을 참조하십시오. </li><li> CO2 패드의 위에, 파리를 표현하는 TAP의 유전자에서 20 성인 머리를 해부하고 각 용해 버퍼 상태를 테스트하기 위해 1.5 ML 튜브에서 그들을 수집 # 5 집게를 사용합니다. </li><li> 튜브에 100 ㎕ 시험 용해 버퍼를 추가하고 또 다른 100 ㎕ 테스트 버퍼를 추가 한 다음, 최대 쓰다듬어에 의해 플라스틱 유 봉 아래로 머리를 균질화. </li><li> 10 분 (4 ℃)를 위해 21,500 XG에서 헤드 해물 스핀, 원심 분리 후 상등액과 펠렛을 분리합니다. 각각 상층 액의 30 UL에 펠릿 10 UL 배 SDS 로딩 버퍼에 25 UL 2X SDS 로딩 버퍼를 추가합니다. </li><li> 5 분 동안 두 개의 샘플을 삶아 나란히 그들에게 분석측 PAP 항체 SDS-PAGE 이후 서양의 얼룩을 사용하여. 펠릿 VS 상청액 TAP-단백질 수준의 비율에 의해 용해도를 결정한다. </li></ol></li><li> 큰 규모의 샘플을 준비 <ol><li> 확장 및 성인 파리를 수집합니다. <ol><li> 병 neuronal-GAL4/UAS-TAP-transgene 주식을 확장하고 누적 250 병까지 3 일마다이 컬렉션에 사용되는 병을 플립. </li><li> 수집 1~3일 오래된 성인 50 ML 원뿔 튜브에 파리, 즉시 냉동이 파리에 액체 질소에 튜브를 넣어. -80 ° C 냉동고에 파리를 저장합니다. 파리의 부피가 50 ㎖ 튜브의 2 / 3를 초과하지 않아야합니다. </li></ol></li><li> (분말 드라이 아이스의 상단에이 단계를 수행) 플라이 헤드를 수집합니다. <ol><li> prechilled 체 및 -80 ° C 냉동고에서 박격포와 유봉을 가지고 이상적으로 큰 얼음 양동이 안에, 드라이 아이스에 넣어. 일에 제 25 호 두 개의 미국 표준 시험 체 스택전자의 상단과 하단에있는 제 40 호. </li><li> 얼어 붙은 파리를 꺼내 액체 질소에서 그들을 드롭하고 약 10 분 동안 거기에 파리를 유지합니다. 소용돌이 또는 몸에서 머리, 다리, 날개를 깨고 적극적으로 튜브를 흔들. </li><li> 맨 체에 혼합물을 부어 함께 두 체를 누른 상태에서 적극적으로 자체를 흔들. 체질 후, 몸은, 상단 체에 머물 머리를 바닥 체에 유지됩니다 비행, 다리, 날개, 및 다른 파편은 드라이 아이스로 떨어질 것입니다. 두 체를 분리하고 조심스럽게 차가운 박격포에 비행 머리를 전송할 수 있습니다. </li></ol></li><li> 플라이 헤드를 균질화 <ol><li> 드라이 아이스 위에, 얼음에 prechilled 된 15 ㎖의 유리 다운스 조직 분쇄기로 가루를 전송 한 후, 분말 입자에 박격포와 유 봉 머리를 갈기. </li><li> 헤드 시료 그것을 붓고 전후 분쇄기의 무게를 측정 한 후 얼마 헤드 SAMP보기 계산할르 무게. 플라이 헤드 6-15그램의 총 단백질의 발현 수준에 따라 조정 TAP 각 실험에 대해 충분할 것이다. 유봉 아래로 이동하는 것이 쉬운 때까지 가루에 얼음처럼 차가운 균질화 버퍼 15 ㎖ (단계 2.2에 최적화 된 용해 버퍼)를 추가 한 다음 큰 간극 유 봉 치기. 항상 얼음에 유리 분쇄기를 유지합니다. </li></ol></li><li> TAP의 상층 액을 준비 <ol><li> ~ 50,000 XG (4 ℃)에서 20 분 동안 고속 원심 분리기 튜브와 스핀에 균질를 전송합니다. 새로운 고속 원심 분리 관에 뜨는을 전송하고 원심 분리를 한 번 더 반복합니다. </li><li> 초원 심 분리기 튜브에 뜨는을 전송하고 더 뜨는을 취소 40 분 ~ 25 XG에 스핀을 수행합니다. 상등액을 원심 분리 후 탠덤 친화 정제 과정에 대한 준비가되어 있습니다. </li></ol></li></ol></li></ol> 3. TAP 정화 </p&gt; 다음 섹션은 Séraphin 실험실 TAP에서 파생 된 protocol 12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf ) <ol><li> IgG의 구슬 친화력 정화를 수행 <ol><li> 샘플을 원심 분리하는 동안의 IgG 세 파로스 비드를 준비합니다. 10 ㎖ 차가운 IgG를 세척 버퍼 15 ML 팔콘 튜브에 400 ㎕의 IgG의 구슬 배를 씻으십시오. 각 세척을 위해, 2 분 동안 부드럽게 튜브를 흔들어 다음 다른 2 분 동안 1,000 XG에서 비즈를 스핀 다운. 제 워쉬의 끝에서, 버퍼를 제거하고 튜브 만 비즈를 남긴다. </li><li> 조심스럽게 IgG의 구슬을 포함하는 15 ML 튜브에 삭제 상층 액 (~ 15 ㎖)에 전송합니다. 2 시간 동안 nutator에 4 º C에서 구슬과 뇌 해물 믹스를 품어. </li><li> 냉장실에 약 15 ml의 총 부피와 깨끗하고 빈 마이크로 열을 설정합니다. 꾸준히 열에 믹스를 부어 IgG의 비드 혼합물을로드, 트랩을하지 않도록 노력공기는 열을 내부에 거품. 구슬이 천천히 중력 흐름에 의해 배출 열 버퍼에 정착 할 수 있습니다. </li><li> 뇌 해물 모두가 해결의 IgG 칼럼을 통해 유입 된 후 차가운 IgG의 세척 완충액 10 ㎖로 철저하게 열을 씻으십시오. 세척의 배를 반복합니다. 참고 : 공기 비드를 건조하게하지 마십시오. </li></ol></li><li> TEV 절단을 수행 <ol><li> 세 번째 세척 후, 10 ㎖ TEV 절단 버퍼를 사용하여 열을 다시 씻는다. 이 단계는 TEV 절단 용 미끼 복잡한 sequestrates IgG의 구슬을 준비합니다. </li><li> TEV 버퍼의 마지막 한 방울은 밖으로 물방울의 흐름을 차단하는 열의 하단에 모자를 쓰고, 열 TEV 효소의 130 단위를 함유하는 1.3 ml의 TEV 버퍼를 추가 한 다음 안전하게의 상단 캡에 대한되기 직전에 열. 열이 양쪽에 잘 밀봉되어 있는지 확인합니다. </li><li> TEV 효소가 TEV 사이트에서 펩티드를 절단 및 단백질 복잡한 WHI을 해제 할 수 있도록 2 시간 동안 18 º C의 열을 회전르 단백질 뒤에 IgG의 세파 로스 구슬에 결합 도메인 펩타이드를 떠나. </li></ol></li><li> 칼 모듈 린 구슬 친화력 정화를 수행 <ol><li> IgG의 구슬이 TEV 효소 배양하는 동안 칼 모듈 린의 구슬을 준비합니다. 버퍼를 바인딩 차가운 칼 모듈 린의 10 ㎖로, 15 ㎖의 팔콘 튜브의 3 배의 각 시간을 200 ㎕의 칼 모듈 린의 구슬을 씻으십시오. 각 세척을 위해, 부드럽게 nutator에 2 분 동안 튜브를 흔들어 다음 2 분 동안 1,000 XG에서 비즈를 스핀 다운. 세 번째 세척의 끝에서, 모든 버퍼를 꺼내 튜브 만 구슬을 떠난다. </li><li> TEV 배양 (단계 3.2.3)의 끝에서 다시 차가운 방에 IgG의 열을 반환하고 똑바로 설정합니다. 구슬이 10 분 동안 정착하자. </li><li> 상단 캡 다음 하단 캡을 제거하고 15 ML 팔콘 튜브에 1.3 ㎖의 TEV 절단 제품을 수집합니다. 버퍼가 완전히 비워집니다. 열 죽은 볼륨을 밀어 열을 추가로 200 ㎕의 TEV 버퍼를 추가, F를 수집같은 튜브 낮은 아웃. </li><li> 상기 수집 된 1.5 ㎖의 TEV 절단 제품에 버퍼와 4.5 ㎕의 1 M 염화칼슘을 결합 칼 모듈 린의 4.5 ML을 추가합니다. 염화칼슘은 TEV 버퍼에 EDTA를 적정하는 역할을한다. 칼 모듈 린의 구슬이 들어있는 튜브에 6 ml의 혼합물을 전송하고 1 시간 동안 nutator에 4 º C에서 튜브를 회전합니다. </li><li> 냉장실에 약 10 ml의 총 부피와 다른 깨끗하고 빈 마이크로 열을 설정합니다. 열에 칼 모듈 린 비드 혼합물을로드하고 중력에 의해 배출 할 수 있습니다. </li><li> 모든 솔루션이 정착 칼 모듈 린의 칼럼을 통해 유입 된 경우, 버퍼를 바인딩 차가운 칼 모듈 린의 10 ㎖로 열 두 번, 각각의 세척. 참고 : 칼 모듈 린의 구슬을 방해 방지하고 세척하는 동안 가능한 한 평면 구슬의 표면을 유지하려고합니다. </li></ol></li><li> 칼 모듈 린 열에서 미끼 복잡한을 용출. 마우스 오른쪽 단추로 세척 한 후, 200 ㎕의 차가운 Calmodu 다섯 분수와 칼 모듈 린 열을 용출린 용출 버퍼. 각 부분의 경우, 부드럽게 열 용출 버퍼 200 μl를 추가하고 표시 1.5 ㎖의 에펜 도르프 튜브 용출액을 수집합니다. 이 배를 반복합니다. </li><li> SDS-PAGE에 의해 단백질 복합체를 분석 <ol><li> 다섯 분수 (약 30 μL)의 각각에서 작은 나누어지는을 가지고 SDS 로딩 버퍼를 추가합니다. 5 분 동안 샘플을 삶아 구배 (4 % -15 %) SDS-PAGE 젤에있는 단백질 분자 마커와 시료를 나란히로드합니다. </li><li> 샘플이 완전히 겔에서 해결 한 후 전기 영동을 중단하고 13 얼룩 &#39;파란은&#39;같은 G-250 기반의 민감한 콜로이드 쿠마시 염색 절차 젤을 얼룩. 그것은 이후의 질량 분석 분석과 완벽하게 호환되지 않기 때문에 염색은 바람직 선택 사항이지만이 아닙니다. 이러한 정제 된 단백질 복합체의 분자 신원을 폭로하기위한 질량 분석 등의 추가 분석을 위해 -80 ° C의 냉동고에 용출액의 나머지 부분을 저장합니다. SE전자 토론. </li></ol></li></ol>

<ol>
	<li>Rigaut, G., 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+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat. Biotechnol. 17, 1030-1032 (1999).</li><li>Collins, S. R., 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=Toward+a+comprehensive+atlas+of+the+physical+interactome+of+Saccharomyces+cerevisiae.">Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.</a> Mol. Cell. Proteomics. 6, 439-450 (1074).</li><li>Gavin, A. 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=Proteome+survey+reveals+modularity+of+the+yeast+cell+machinery.">Proteome survey reveals modularity of the yeast cell machinery.</a> Nature. 440, 631-636 (2006).</li><li>Krogan, N. J., 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=Global+landscape+of+protein+complexes+in+the+yeast+Saccharomyces+cerevisiae.">Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.</a> Nature. 440, 637-643 (2006).</li><li>Forler, D., 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=An+efficient+protein+complex+purification+method+for+functional+proteomics+in+higher+eukaryotes.">An efficient protein complex purification method for functional proteomics in higher eukaryotes.</a> Nat. Biotechnol. 21, 89-92 (2003).</li><li>Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+complexes+in+Drosophila+with+tandem+affinity+purification-mass+spectrometry.">Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.</a> Dev. Dyn. 232, 827-834 (2005).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+technology:+an+overview.">The tandem affinity purification technology: an overview.</a> Biotechnol. Lett. 33, 1487-1499 (2011).</li><li>Volkel, P., Le Faou, P., Angrand, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+proteomics:+characterization+of+protein+complexes+using+tandem+affinity+purification-mass+spectrometry.">Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry.</a> Biochem. Soc. Trans. 38, 883-887 (2010).</li><li>Xu, X., 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=The+tandem+affinity+purification+method:+an+efficient+system+for+protein+complex+purification+and+protein+interaction+identification.">The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.</a> Protein Expr. Purif. 72, 149-156 (2010).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Bachmann, A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+P-element+transposons+to+generate+transgenic+flies.">The use of P-element transposons to generate transgenic flies.</a> Methods Mol. Biol. 420, 61-77 (2008).</li><li>Puig, O., 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=The+tandem+affinity+purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 (2001).</li><li>Candiano, G., 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=Blue+silver:+a+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25, 1327-1333 (2004).</li><li>Tian, X., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ubiquitin-mediated+pathways+in+regulating+synaptic+development,+axonal+degeneration+and+regeneration:+insights+from+fly+and+worm.">The role of ubiquitin-mediated pathways in regulating synaptic development, axonal degeneration and regeneration: insights from fly and worm.</a> J. Physiol. , (2013).</li><li>Wu, C., Wairkar, Y. P., Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highwire+function+at+the+Drosophila+neuromuscular+junction:+spatial,+structural,+and+temporal+requirements.">Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.</a> J. Neurosci. 25, 9557-9566 (2005).</li><li>Wu, C., Daniels, R. W., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DFsn+collaborates+with+Highwire+to+down-regulate+the+Wallenda/DLK+kinase+and+restrain+synaptic+terminal+growth.">DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth.</a> Neural Dev. 2, 16 (2007).</li><li>Tian, X., Li, J., Valakh, V., DiAntonio, A., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Rae1+controls+the+abundance+of+the+ubiquitin+ligase+Highwire+in+post-mitotic+neurons.">Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons.</a> Nat. Neurosci. 14, 1267-1275 (2011).</li><li>Burckstummer, T., 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=An+efficient+tandem+affinity+purification+procedure+for+interaction+proteomics+in+mammalian+cells.">An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.</a> Nat. Methods. 3, 1013-1019 (2006).</li><li>Kyriakakis, P., Tipping, M., Abed, L., Veraksa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+in+Drosophila:+the+advantages+of+the+GS-TAP+system.">Tandem affinity purification in Drosophila: the advantages of the GS-TAP system.</a> Fly. 2, 229-235 (2008).</li><li>Bailey, D., Urena, L., Thorne, L., Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+interacting+partners+using+tandem+affinity+purification.">Identification of protein interacting partners using tandem affinity purification.</a> J. Vis. Exp. (60), e3643 (2012).</li><li>Wu, Y., 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+Drosophila+model+for+Angelman+syndrome.">A Drosophila model for Angelman syndrome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 12399-12404 (2008).</li><li>Liao, L., McClatchy, D. B., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+proteomics+in+neuroscience.">Shotgun proteomics in neuroscience.</a> Neuron. 63, 12-26 (2009).</li></ol>

저자가 공개하는 게 없다.

탠덤 선호도 정화 (TAP) 메서드는 두 개의 독립적 인 친 화성 정제 단계를 통해 분리 및 단백질 단지의 농축을 할 수있는 듀얼 정화 프로토콜을 제공합니다. TAP 태그의 디자인이 프로토콜에 제시되어 무엇에 한정되지 않고 버퍼 조건을 적절하게 조정하는 경우, 다른 단백질 결합 도메인 및 모티프도 적용 할 수 있습니다. 다른 TAP 태그의 좋은 예는 GS-TAP 태그, G 단백질의 조합 배양 포유 동물 세포 라...

특정 생물 학적 과정을 중재하는 분자 경로 또는 네트워크를 정의하는 것은 생물 의학 연구의 궁극적 인 목표 중 하나입니다. 비행 유전 학자, 특히 병렬로 함께 작동, 또는 상류 또는 그 유전자의 하류 요인을 식별하는 유전자 화면 (증강 및 회로 화면을 모두) 수정, 앞으로 유전학에 크게 의존했다. 그러나, 앞으로 유전학 화면은 종종 변이 된 경우, 그 손실 함수의 유일한 득점하기 어려운 미묘한 결함의 원인이 기능 중복 및 보상 초기 발달 단계에서 치사, 또는 유전자를 일으키는 원인이되는, 필수 유전자를 확인하기 위해 실패합니다. 이러한 어려움을 극복하는 방법 중 하나는 직접 단백질 - 단백질 상호 작용에 대한 화면이다. 이상 10 년 동안 등 효모 두 하이브리드, 파지 디스플레이, 화학 가교, 공동 IP, 탠덤 선호도 정화 (TAP), 등의 생화학 적 방법의 성장 목록. 단백질을 조사하기 위해 사용되어왔다- 단백질 상호 작용. 각각의 방법은 장점과 민감도와 특이도에 관해서 약점 자체 집합이 있습니다. 그 중에서도, TAP 방식은, 거의 생리적 조건 하에서 물리적 상호 작용의 검출을 허용 특이성 및 일관성이 유지되고 3,4 분석 높은 처리량을 확장 할 수있는 능력을 포함한다. TAP 방법은 원래 Rigautand 동료 (1)에 의해 효모에 개발되었다. 이 방법에서는, 그 단백질은 TAP 태그로 표현된다. 단백질의 IgG에 결합 도메인과 칼 모듈 린 결합 도메인 : TAP 태그는 두 개의 독립적 인 친 화성 결합 도메인을 항구. 두 도메인은 TEV (담배 식각 바이러스) 절단 부위로 구분됩니다. 선호도 않는 정화의 두 개의 독립적 인 라운드가 충분히 특이 현상이 바인딩을 줄이고 특정 바인딩 1을 풍성하게하는 그런 조합 할 수 있습니다. 이것은 예를 들어, TAP 방식은 매우 강력하다 메 톡시외래 단백질을 과발현하는 것은 일반적으로 그 내생 대응 복잡한하지 않는 단백질과 연결하기가 더 많은 경향이 만들 수 있지만, 특정 단백질의 생체 내 상호 작용을 식별하기 위해 개발. 개발 이후, TAP 방법은 세포 배양 기반 시스템 5,6 및 기타 생체 내 모델 시스템 6-9 포함하여 많은 다른 시스템에 적용되었습니다. 여기에서 우리는 초파리의 TAP 방법의 적응을 설명합니다. 우리는 먼저 그 유전자의 N-또는 C-말단 하나에 TAP 태그의 복제 및 융합을 촉진하기 위해 pUAST-NTAP 및 pUAST-CTAP 벡터를 생성합니다. UAS-TAP-태그 된 트랜스이어서 신경 GAL4 드라이버 (10)의 제어하에 신경계에서 발현된다. 다음으로, 성인 플라이 헤드의 다수의 신경 세포의 높은 함량을 가지고 사이즈 차이에 기초하여 고정한 후 다른 신체 부위로부터 분리하기 쉽고, 이는 모아질 것이다. 성인 머리 균질화 지울 B입니다Y 연속 centrifugations, 상층 액은 아래에 설명 TAP 절차의 적용을받습니다.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">U.S.A. standard test sieve No. 25</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-18&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Kontes Dounce Tissue Grinders 15 ml</td>
			<td>Kimble Chase</td>
			<td>885300-0015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;"></td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG sepharose beads</td>
			<td>Pharmacia</td>
			<td style="width:119px;">17-0969-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.7 cm x 20 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4721</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.5 cm x 15 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4716</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calmodulin beads</td>
			<td style="width:104px;">Stratagene</td>
			<td style="width:119px;">214303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coors Mortar and Pestle</td>
			<td style="width:104px;">CoorsTek</td>
			<td>60311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AcTEV Protease</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:104px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Mix</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

초파리 melanogaster의 (초파리)를 사용하여 실시 유전 화면은 생명 과학의 발전에 많은 이정표 발견을 만들었습니다. 그러나, 유전자 분석에서 얻은 지식을 연장 겨냥 생화학 스크린의 사용은 단지 최근 탐구 하였다. 여기에서 우리는 단백질 복합체를 정화 할 수있는 방법을 설명하는 성인 플라이 헤드 관심의 단백질과 동료. 이 방법은 생체 내에서 비행 신경에있는 탠덤 선호도 정화 (TAP) 태그와 융합 미끼 단백질을 발현하는 초파리 GAL4/UAS 시스템을 활용하고 원래 년에 설립 된 것과 유사한 TAP 절차를 사용하여 정화의 두 라운드를 구현 상호 작용하는 단백질 복합체를 정제하는 효모 1. 이 절차의 끝에서, 다중 단백질 복합체의 혼합물을 그 분자 신원 질량 분석법에 의해 결정될 수있다 얻어진다. 후보 단백질의 유효성 검사는 R 도움이됩니다esource과 파리에서 손실의 기능 연구를 수행의 용이성. 유사한 접근법이 다른 플라이 조직에 적용될 수있다. 우리는 비행 모델 시스템에서 유전자 조작이 단백질 체학 접근 방식의 조합은 신경 생물학 분야와 넘어 근본적인 문제를 다루기위한 엄청난 잠재력을 보유하고 있다고 생각합니다.

여기에서 우리는 즉시 뇌에 Highwire 상호 작용하는 단백질을 식별하는 우리의 노력을 보여줍니다. Highwire (HIW)과 척추 동물과 무척추 동물 상동 신경계 (14)의 개발 및 수리를 조절 거대한 유비퀴틴 리가 제입니다. 그들은 고도의 보존 기능 영역의 번호를 공유 할 수 있습니다. 그러나, 그들의 분자의 행동은 완전히 명확하지 않습니다. 웜에 수행 한 작업은, 비행 및 마우스가 현재 작업 모델?...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP at JoVE.com

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

초파리는하지만 깊이있는 생화학 적 분석의 적합성에 대한, 강력한 유전자 조작으로 유명합니다. 여기에서 우리는 파리의 뇌에서 관심의 단백질의 상호 작용 파트너를 식별 할 수있는 TAP 기반의 절차를 제시한다. 이 절차는 잠재적으로 연구의 새로운 길을 초래할 수 있습니다.

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. ...

identifying-protein-protein-interaction-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

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

13.8K 조회수.  Louisiana State University Health Sciences Center.  초파리는하지만 깊이있는 생화학 적 분석의 적합성에 대한, 강력한 유전자 조작으로 유명합니다. 여기에서 우리는 파리의 뇌에서 관심의 단백질의 상호 작용 파트너를 식별 할 수있는 TAP 기반의 절차를 제시한다. 이 절차는 잠재적으로 연구의 새로운 길을 초래할 수 있습니다. 

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

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

작은 Bispecific 선호도 태그에 의해 용이 직교 단백질 정제

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

연구

생물학

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 ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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 ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

미세 규모 Thermophoresis에 의한 결합력 결정 단백질 정제 - 무료 방법

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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

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

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

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

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

살아있는 세포의 단백질 조각 보완 분석 (PCA)에 의해 게놈 전체 단백질 - 단백질 상호 작용 상영

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...