Name: identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

We thank Dr. Ben Nacev for advice on design of the photoaffinity labeling protocol, Dr. Wei Shi for synthesizing the itraconazole photoaffinity probe, Dr. Yongjun Dang for advice on affinity pull-down experiments, and other members of the J.O.L. laboratory for helpful comments and support. This work was supported in part by a PhRMA Foundation Fellowship in Pharmacology/Toxicology (to S.A.H.); National Cancer Institute Grant R01CA184103; the Flight Attendant Medical Research Institute; Prostate Cancer Foundation (J.O.L.); and the Johns Hopkins Institute for Clinical and Translational Research, which is funded in part by Grant UL1 TR 001079 from the National Center for Advancing Translational Sciences (NCATS).

참고 :이 프로토콜은 살아있는 세포에 사용 맥키와 톤턴 (10)에서 적응했다. 배양 된 세포의 1. 준비 <ol><li> (아래 참조) 원하는 샘플 수에 대한 멸균 6 cm 세포 배양 접시를 준비합니다. 주 : 셀들 중 하나 접시가 처리 조건에 따라 사용되지만, 더 많은 단백질이 2 또는 3 요리 상태별로 준비하고, UV 조사 후 결합 될 수있다 요구되는 경우. <ol><li> 세포의 최소 3 요리를 준비; DMSO 만 (D) 프로브 처리 만 (P)과 프로브 + 경쟁 (C)와 음성 대조군. </li><li> 선택적으로, 아니 UV 컨트롤로 4 번째 접시, 프로브로 치료하지만 자외선에 노출되지 할 준비를합니다. 주 : 여러 경쟁 분자는 관심 소분자 다른 유사체로서 시험 할 경우, 필요에 따라 추가로 경쟁 판을 준비한다. </li></ol></li><li> 4 ml의 배양 배지에서 배양 접시에 HEK293T 세포를 추가합니다. 350 만 C를 사용하여접시 당 ELL 학생. 주 : 셀은 실험 시작시 약 100 % 컨 플루 언트되도록 세포의 수가 최대 단백질 수율을 달성하기 위해, 각 세포 유형에 대해 결정되어야한다. HUVEC를 들어, 접시 당 0,300,000 세포를 사용합니다. 좋은 근사 합류 15cm 접시에 세포의 1/10이다. HEK293T 세포에서 배양한다 둘 베코 변형 이글스 중간 10 % 소 태아 혈청 (FBS), 1 % 페니실린 / 스트렙토 마이신이 보충. </li><li> 문화 인큐베이터로 세포를 반환 (37 ° C, 5 % CO 2) 하룻밤. </li></ol> 세포 및 광 가교 2. 치료 <ol><li> 다음 날, 미리 나누어 1.5 ml의 마이크로 원심 튜브에 다음 약물 : <ol><li> 첫 번째 치료를 위해, 2 관 1 관 10 mM의 경쟁자 (즉, 수정되지 않은 모 화합물)의 4 μL에 DMSO의 4 μl를 추가합니다. </li><li> 두 번째 치료를 위해, 1 관 DMSO의 16 μl를 50 &amp; 16 μl를 추가# 181; 2 관에 M의 photoaffinity 프로브. 주 : 최적 농도는 다른 프로브 (설명을 참조)에 대해 상이 할 수 있지만, 200 ~ 500 nm의 사이에서 좋은 출발점 (여기에서는 200 nm의 사용)이다. 경쟁자의 농도는 프로브 최소 20 배 (여기에 우리가 10 μM 사용)의 초과한다. DMSO의 최종 농도는 모든 판에서 동일해야하며, 0.5 % (20 μL 볼륨)를 초과하지 않아야합니다. </li></ol></li><li> 세포 배양 후드로 셀 플레이트를 가지고 다음과 같이 단계 2.1.1에서 프리 분주 약물을 추가 배양액 대기음 1ml를 상기 배지에서 미리 분주 약물을 재현 탁하고, 가볍게 플레이트 강하로 다시 추가 슬기로운. DMSO를 (D) 플레이트와 프로브 (P) 판에 DMSO를 추가하고, 경쟁 (C) 판에 참가자를 추가합니다. 30 분 동안 배양 인큐베이터에 접시를 돌려줍니다. </li><li> 흐리게 문화 후드에서 조명과 함께, 조사 단계 2.1.2에서 미리 분주 프로브를 추가 (P)과 경쟁 (C) 판과 추가상기와 DMSO (D) 판 DMSO. 1 시간 동안 배양 인큐베이터에 접시를 돌려줍니다. </li><li> 배양하는 동안, 다음 항목을 준비; 판을 모두 들어갈 수있는 얼음 트레이; 빙냉 용해 완충액 (PBS [pH를 8.5] + 1X 프로테아제 억제제 칵테일 [완전 EDTA없는 물로 이루어진 50X 원액으로부터 희석] 200 μL / 플레이트); 마이크로 원심 튜브를 얼음에, 다른 처리 조건들 각각에 대해 D, P, 또는 C로 표시. </li><li> 1 시간 배양이 끝나기 전에 15 분, 추운 방에 UV 램프 (365 나노 미터)를 설정하고 전구를 따뜻하게 전원을 켭니다. </li><li> 프로브와 1 시간 배양 한 후, 얼음에 요리를 놓습니다. 여분의 프로브를 제거하고 부드럽게 5 ml의 얼음처럼 차가운 PBS (PH 7.4)로 세포를 씻으십시오. 4 ml의 빙냉 PBS로 세포를 다시하면-커버. </li><li> 램프의 발열을 최소화하기 위해 얼음 팩의 상단에 UV 램프에서 3cm를 중심으로 세포의 요리를 놓습니다. 3 분 동안 조사. 얼음 트레이에 접시를 제거하고 모든 샘플에 대해 반복합니다. </li><li> 에이따고 조사는 세포에서 PBS를 대기음하고 각 플레이트에 단백질 분해 효소 억제제와 얼음처럼 차가운 PBS (PH 8.5)의 200 μl를 추가합니다. 얼음에 미리 라벨링 마이크로 원심 튜브 고무 스크레이퍼 및 전송을 사용하여 플레이트로부터 세포를 분리. </li><li> (샘플 250 μL에 10 % SDS 10 μL)에 0.4 %의 최종 농도로 SDS를 추가한다. 주의 : SDS 분말은 위험합니다. 코와 입을 통해 마스크를 착용하여 SDS 분말을 흡입하지 마십시오. </li><li> 10 펄스를 Lyse (출력 1, 듀​​티 사이클 30 %)를위한 현탁액을 초음파 처리하고 10 펄스의 두 번째 라운드 전에 1 분 동안 얼음에 부화하여 세포. </li><li> 필요한 경우 현탁액 2 μL를 제거하고 전체 세포 용해를 확인하기 위해 광학 현미경 검사. </li><li> 세포 용해를 완료하고 모든 단백질 변성을 5 분 동안 95 ℃로 설정 한 핫 플레이트에 샘플을 끓인다. </li><li> 이러한 번째로, 분광 세제 대응의 단백질 분석을 이용하여 각 샘플의 단백질 농도를 측정750 nm에서 흡광도를 측정하도록 설정된 분광 광도계 제조업체의 지시에 따라 전자 DC 단백질 분석 19. </li><li> 필요 PBS pH를 8.5 + 0.4 % SDS를 첨가하여 2.5 ㎎ / ㎖로 단백질 농도를 정상화 (또는 농도 인 경우보다 낮은 2.5 ㎎을 / ㎖, 가장 낮은 단백질 농도 시료 정상화) (예를 들어, 샘플 5 있다면 250 μL의 ㎎ / ㎖는) 2.5 mg / ml의 최종 농도를 얻기 위해 PBS pH를 8.5 + 0.4 % SDS 250 μL를 추가한다. </li></ol> 레이블이 단백질의 시각화를 클릭 화학에 의한 형광 태그 3. 첨부 파일 <ol><li> 새의 microcentrifuge 튜브에 세포 용 해물 및 전송의 40 μl를 제거합니다. 비오틴 - 아 지드 (4 단계)과의 반응에 대한 나머지 해물을 유지합니다. </li><li> 순서대로 다음과 같은 시약을 추가 : 0.2 μL의 불소 - 아 지드 (DMSO의 1 mM의 원액), 0.58 μL의 TCEP (100 mM의 주식을 물에 수산화 나트륨을 첨가 4 당량, 신선한 준비), 및 3.38 μl를 TBTA (1.4 7 밀리미터 재고 : DMSO에 t- 부탄올의 1 비율). 소용돌이는 혼합한다. </li><li> 반응을 시작합니다 1.14 μl를 -5H 2 O (물에 50 mM)을 CuSO 4를 추가합니다. 간단히 와동과 어둠 속에서 30 분 동안 실온에서 배양한다. </li><li> 반응을 종결 배 SDS 샘플 버퍼의 50 μl를 추가합니다. 이 시점에서 하룻밤 필요한 경우 -20 ° C에서 최상의 결과를, 또는 상점에 대한 SDS-PAGE 겔 (20)에 직접 샘플을 실행합니다. </li><li> 샘플을 동결하면 겔 상에 로딩하기 전에 95 ℃에서 5 분 동안 가열한다. 주 : 목적 단백질의 분자량은 일반적으로이 단계에서 공지되어 있지 않기 때문에, 그것은 다른 아크릴 아마이드 농도가이 겔을 실행하는 것이 바람직하다 (즉, 8 % 및 15 %), 또는 넓은 분자량 범위 구배 겔 (즉, 4-15%)는 전체 분자량 범위를 확인합니다. </li><li> 염료 프론트 겔의 끝에 도달하면, 초과 unreacte 모두를 위해 추가로 5 분 동안 겔을 계속 실행할(D) 불소 지드 완전히 겔을 종료했다. </li><li> DDH 2 O와 컨테이너에 젤을 제거하고 멀리 모든 초과 불소 - 아 지드를 씻어, 부드러운 교반과 함께 10 분 동안 품어. </li><li> 유리 기판 상에 겔을 위치시키고 제조자의 지시에 따른 형광 태풍 겔 스캐너를 사용하여 겔을 검사한다. </li></ol> 레이블이 단백질의 선호도 정화를 클릭 화학에 의한 비오틴 태그 4. 첨부 파일 <ol><li> 단계 3.1에서 나머지 해물 중에서 후 정규화 2.5 ㎎ / ㎖ 얻어진 샘플 볼륨은 500, 550, 600 μL를 있으면 모든 샘플은 (예를 들어 동일한 양이되도록 단백질 정규화 이후 최대 크기를 사용하여, (500)을 사용하여 각각의 μL). </li><li> PBS (PH 7.4) 50 μL에 고용량 스트렙 아가 로스 비드, 예비 세척 된 배를 추가하여 용 해물을 미리 투명한. 회전 4 ° C에서 1 시간을 품어. </li><li> 3 분 동안 1,000 XG에서 원심 분리하여 구슬을 펠렛. t을 제거그는 얼음에 새로운 microcentrifuge 관에 뜨는와 구슬을 버린다. </li><li> &quot;입력&quot;이라는 새로운 튜브에 DMSO 샘플의 1 ~ 2 %를 제거합니다. -20 ° C에서 2 배 SDS 샘플 버퍼와 저장소의 동등한 볼륨을 추가합니다. </li><li> 해물의 500 μL 당, 다음과 같은 시약을 추가 : 1.38 μL 비오틴 - 아 지드 (DMSO에서 10 밀리미터 주), 5.5 μL TCEP, 32.5 μL의 TBTA합니다. 소용돌이는 혼합한다. </li><li> 11 μl를 간단히 해물과 소용돌이의 500 μL 당 -5H 2 O CuSO 4를 추가합니다. 실온에서 30 분 동안 인큐베이션. </li><li> -20 ° C로 냉각 아세톤 4 샘플 볼륨을 추가합니다. 샘플 및 -80 ° C에서 밤새 품어 소용돌이 완전히 단백질을 침전과 미 반응 비오틴 - 아 지드를 제거합니다. </li><li> 원심 분리기는 4 ° C에서 15 분 동안 17,000 XG에 샘플을 침전 된 단백질을 펠렛합니다. </li><li> 완전히 뜨는을 대기음, 150 μl의 PBS (산도 7.4) + 1 % SDS에서 초음파에 의해 단백질을 resolubilize. </li><li&gt; 0.2 %로 SDS의 농도를 희석하기 위해 PBS (산도 7.4) 600 μl를 추가합니다. </li><li> 30 μl를 미리 씻어 고용량 스트렙 타비 아가로 오스 비즈에 샘플을 추가하고 회전 4 ° C에서 1 시간을 품어. </li><li> 3 분 동안 1,000 XG에서 원심 분리하여 구슬을 펠렛. 언 바운드 단백질 및 폐기를 포함 뜨는을 대기음. </li><li> 구슬로 세척 완충액 (400 mM의 염화나트륨, 50 mM 트리스, 0.2 % SDS, pH를 7.4) 1 ㎖를 추가하고 회전 실온에서 5 분 품어. </li><li> 반복 4.12 및 4.13 3 번 단계를 반복합니다. </li><li> 구슬에서 완전히 세척 버퍼를 대기음, 그리고 배 SDS 샘플 버퍼의 30 μl를 추가합니다. </li><li> 비즈에서 단백질을 분리하기 위해 95 ° C 열 블록에 5 분 동안 품어. </li><li> 실온에서 13,000 × g으로 1 분간에 비드를 원심 분리기. 주 :이 시점에서, 샘플을 SDS-PAGE를 실행할 준비가 될 때까지 -20 ℃에서 저장 될 수있다. 시료를 냉동하는 경우, 사용하기 전에 95 ℃에서 5 분 동안 가열한다. </li><li> 면밀히비드 오프 단백질을 포함하는 시료 완충액을 피펫 및 SDS-PAGE (20) 상에로드 (아래 중요한 고려 사항 참조). 비드 리 보일 필요 이상 동안 저장 될 수있다. 주 : 실버 염색에 의한 단백질의 검출을 위해, 더 나은 결과가 1 mm 두께의 겔을 사용하여 얻어진다. 밴드는 질량 분석 (MS) 분석을위한 실버 스테인드 겔에서 절단 할 경우, 멸균 솔루션에서 신선한 모든 시약을 준비하고 겔에 샘플들 사이에서 잘 빈 둡니다. 그 배경 단백질 감산 될 수 있도록, 프로브로부터 절단 차선마다 대역 병렬 슬라이스는 DMSO 차선에서 촬영한다. 웨스턴 블롯에 의해 단백질의 확인을위한 ID, 상기 SDS-PAGE 젤을 실행하는 경우에도 단계 4.4로부터의 입력 샘플을 로딩하는 것이 중요하다. </li><li> 표적 단백질 (들)을 감지하는 실버 염색 (21) 또는 웨스턴 블롯 (22) 중 하나의 표준 프로토콜을 따르십시오. </li></ol>

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	<li>Schenone, M., Dan&#269;&#236;k, V., Wagner, B. K., Clemons, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+and+mechanism+of+action+in+chemical+biology+and+drug+discovery.">Target identification and mechanism of action in chemical biology and drug discovery.</a> Nat Chem Biol. 9 (4), 232-240 (2013).</li><li>Cook, D., Brown, 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=Lessons+learned+from+the+fate+of+AstraZeneca's+drug+pipeline:+a+five-dimensional+framework.">Lessons learned from the fate of AstraZeneca's drug pipeline: a five-dimensional framework.</a> Nat Rev Drug Discov. 13 (6), 419-431 (2014).</li><li>Titov, D. V., Liu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+protein+targets+of+bioactive+small+molecules.">Identification and validation of protein targets of bioactive small molecules.</a> Bioorg Med Chem. 20 (6), 1902-1909 (2012).</li><li>Jung, H. J., Kwon, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+deconvolution+of+bioactive+small+molecules:+the+heart+of+chemical+biology+and+drug+discovery.">Target deconvolution of bioactive small molecules: the heart of chemical biology and drug discovery.</a> Arch Pharm Res. 38 (9), 1627-1641 (2015).</li><li>Sumranjit, J., Chung, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Target+Characterization+and+Identification+by+Photoaffinity+Probes.">Recent Advances in Target Characterization and Identification by Photoaffinity Probes.</a> Molecules. 18 (9), 10425-10451 (2013).</li><li>Robles, O., Romo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemo-+and+site-selective+derivatizations+of+natural+products+enabling+biological+studies.">Chemo- and site-selective derivatizations of natural products enabling biological studies.</a> Nat Prod Rep. 31 (3), 318-334 (2014).</li><li>Zhou, Q., Gui, 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=Bioconjugation+by+Native+Chemical+Tagging+of+C-H+Bonds.">Bioconjugation by Native Chemical Tagging of C-H Bonds.</a> J Am Chem Soc. 135 (35), (2013).</li><li>Li, Z., Hao, 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=Design+and+Synthesis+of+Minimalist+Terminal+Alkyne-Containing+Diazirine+Photo-Crosslinkers+and+Their+Incorporation+into+Kinase+Inhibitors+for+Cell-+and+Tissue-Based+Proteome+Profiling.">Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and Tissue-Based Proteome Profiling.</a> Angew Chem Int Edit. 52 (33), 8551-8556 (2013).</li><li>Chamni, S., He, Q. L., Dang, Y., Bhat, S., Liu, J. O., Romo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diazo+reagents+with+small+steric+footprints+for+simultaneous+arming/SAR+studies+of+alcohol-containing+natural+products+via+O-H+insertion.">Diazo reagents with small steric footprints for simultaneous arming/SAR studies of alcohol-containing natural products via O-H insertion.</a> ACS chem biol. 6 (11), 1175-1181 (2011).</li><li>Mackinnon, A. L., Taunton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+Identification+by+Diazirine+Photo-Cross-linking+and+Click+Chemistry.">Target Identification by Diazirine Photo-Cross-linking and Click Chemistry.</a> Curr Protoc Chem Biol. 1, 55-73 (2009).</li><li>Smith, E., Collins, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoaffinity+labeling+in+target-+and+binding-site+identification.">Photoaffinity labeling in target- and binding-site identification.</a> Future med chem. 7 (2), 159-183 (2015).</li><li>Huisgen, R., Szeimies, G., M&#246;bius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1.3-Dipolare+Cycloadditionen,+XXXII.+Kinetik+der+Additionen+organischer+Azide+an+CC-Mehrfachbindungen.">1.3-Dipolare Cycloadditionen, XXXII. Kinetik der Additionen organischer Azide an CC-Mehrfachbindungen.</a> Chem Ber. 100 (8), 2494-2507 (1967).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry:+Diverse+Chemical+Function+from+a+Few+Good+Reactions.">Click Chemistry: Diverse Chemical Function from a Few Good Reactions.</a> Angew Chem Int Ed Engl. 40 (11), 2004-2021 (2001).</li><li>Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+huisgen+cycloaddition+process:+copper(I)-catalyzed+regioselective+&amp;#34;ligation&amp;#34;+of+azides+and+terminal+alkynes.">A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective &amp;#34;ligation&amp;#34; of azides and terminal alkynes.</a> Angew Chem Int Ed Engl. 41 (14), 2596-2599 (2002).</li><li>Torn&#248;e, C. W., Christensen, C., Meldal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidotriazoles+on+Solid+Phase:+[1,2,3]-Triazoles+by+Regiospecific+Copper(I)-Catalyzed+1,3-Dipolar+Cycloadditions+of+Terminal+Alkynes+to+Azides.">Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides.</a> J Org Chem. 67 (9), 3057-3064 (2002).</li><li>Head, S. A., Shi, W., 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=Antifungal+drug+itraconazole+targets+VDAC1+to+modulate+the+AMPK/mTOR+signaling+axis+in+endothelial+cells.">Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells.</a> P Natl Acad Sci USA. 112 (52), E7276-E7285 (2015).</li><li>Shi, W., Nacev, B. A., Aftab, B. T., Head, S., Rudin, C. M., Liu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itraconazole+Side+Chain+Analogues:+Structure-Activity+Relationship+Studies+for+Inhibition+of+Endothelial+Cell+Proliferation,+Vascular+Endothelial+Growth+Factor+Receptor+2+(VEGFR2)+Glycosylation,+and+Hedgehog+Signaling.">Itraconazole Side Chain Analogues: Structure-Activity Relationship Studies for Inhibition of Endothelial Cell Proliferation, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, and Hedgehog Signaling.</a> J Med Chem. 54 (20), 7363-7374 (2011).</li><li>Shi, W., Nacev, B. A., Bhat, S., Liu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Absolute+Stereochemistry+on+the+Antiangiogenic+and+Antifungal+Activities+of+Itraconazole.">Impact of Absolute Stereochemistry on the Antiangiogenic and Antifungal Activities of Itraconazole.</a> ACS Med Chem Lett. 1 (4), 155-159 (2010).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> J Biol Chem. 193 (1), 265-275 (1951).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Bioph Res Co. 28 (5), 815-820 (1967).</li><li>Switzer, R. C., Merril, C. R., Shifrin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+silver+stain+for+detecting+proteins+and+peptides+in+polyacrylamide+gels.">A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.</a> Anal Biochem. 98 (1), 231-237 (1979).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> P Natl Acad Sci USA. 76 (9), 4350-4354 (1979).</li></ol>

The authors have nothing to disclose.

약물의 세포 표현형가 알려진 기능에 따라 잠재적 인 대상을 좁힐하는 데 사용되는 하향식, 또는 상향식 (bottom-up), 대상 : 작은 분자의 목표를 확인하는 다른 방법은 크게 두 가지 범주로 분류 할 수 있습니다 화학 물질 또는 유전 적 수단 (3)에 의해 직접적으로 식별됩니다. 하향식 또는 표현형 연구 약물의 영향을 특정 세포 프로세스를 식별 할 수있다 (예를 들면, 전사 / 번역 / DNA ?...

생리 활성 소분자 근본적 셀과 상호 작용하는 하나 이상의 &quot;대상&quot;분자, 가장 일반적으로 단백질의 기능을 변경함으로써 작동한다. 약물 발견에 때 활성 화합물 표현형 선별을 통해 발견되고, 그 화합물의 분자량이 타겟 (들)의 식별은 작용 화합물의 잠재적 부작용의 메커니즘을 이해뿐만 아니라 대한 잠재적으로 발견되지 중요 새로운 생물학 질병 모델을 기본 및 치료제 (1)의 새로운 기계적인 클래스의 개발을위한 방법을 포장. 목표 식별이 치료에 사용하고자하는 약물이 필요하지 않지만, 최근에는 신약 후보 검증 대상이 알려진 경우보다 투자 수익률을 수득 때문에 임상 시험에 성공하고, 가능성이 있음을 점점 인식되고있다 2. 따라서, 작은 식별하는 방법에 대한 관심이 증가되고있다분자 표적 단백질. 고전 목표 식별 실험은 일반적으로 관심의 작은 분자는 수지상에 고정화 비 결합 단백질을 씻어, 나머지 단백질 용출 3를 식별 한 후, 전체 세포 용 해물과 배양 친 화성 정제에 의존한다. 이 기술은 여러 개의 작은 분자들 (4)의 타겟을 식별하기 위해 사용되었지만, 여러 가지 이유로, 범용 대상 ID 방법으로 적합하지 않다. 먼저, 표적 단백질은 작은 분자에 결합하는 능력을 유지하기 위해 세포 용해시의 기본 형태를 유지해야한다. 이것은 종종 그들의 네이티브 환경에서 제거 된 후 구조적인 변화를 거쳐, 또는 단순히 집계 및 용액으로부터 침전 막 단백질, 특히 문제가 될 수 있습니다. 유지하면서 두 번째로, 작은 분자는 화학적으로는 수지에 고정화 될 수있는 방식으로 수정해야표적 단백질에 결합 할 수있는 능력. 이 수지에 고정되면 깊은 결합 포켓 따라서 작은 분자에 액세스 할 수 없게 될 수 있습니다. 셋째, 결합 친화력은 상호 작용이 어려운 낮은 친화력 상호 작용을 확인하기가 세정 단계 동안 유지되도록 충분히 높아야한다. 등의 pH, 이온 농도, 또는 다른 내인성 분자의 존재 넷째, 환경 조건은 셀 내의 공간적으로 다양하고 때로는 약물 표적 상호 작용을 전제되어있다. 따라서, 허용 시행 착오 상당량 필요로 할 수있는 셀의 외부 결합 유지하도록 적절한 조건을 찾아 내기. Photoaffinity 라벨링 소분자와 셀의 기본 문맥 내에서 타겟의 공유 결합을 허용함으로써 이러한 문제를 회피. 오히려 부피가 큰 수지 소분자 고정화보다 분자 대신 두 개의 작은 화학적 작용 g을 설치하도록 수정roups : 빛의 특정 파장의 조사 대상의 단백질에 공유 결합 가교를 할 수있는 광활성 부위 및 표적 단백질을 검출하고이어서 분리 할 수​​있는 리포터 기. 생균 프로브가 공유 결합하여 표적 단백질에 가교 및 프로브 단백질 복합체는 절연 그대로 상기 photoaffinity 프로브로 처리된다. 표적에 결합하는 프로브의 특이성은 모 화합물의 과량의 표적 단백질로 프로브의 결합 거리 경쟁하는 데 사용되는 병렬로 경쟁 실험을 수행함으로써 입증된다. photoaffinity 프로브의 설계 및 합성은 하나의 작은 분자에서 다른 매우 다양하고,이 프로토콜에 포함되지 않습니다; 그러나, 주제에 대한 몇 가지 훌륭한 토론 5-9을 발표했다. 주요 고려 presumab 따라서, 프로브는 모 화합물의 생물학적 활성을 유지한다는 것이다LY는 동일한 타겟 (들)에 대한 결합. 구조 활성 관계 (SAR) 시험은 분자의 일부가 생체 활성의 상실없이 변형 될 수 있는지 결정하기 위해 수행되어야한다. 다른 화학 그룹이 다양한 각각의 장단점이 10 diazirine 페논, 아릴 아 지드를 포함 광활성 가교제,로서 사용되어왔다. 마찬가지로, 프로브 - 결합 단백질을 분리하는 데 사용 된 여러 리포터 태그가있다. 리포터 그룹은 일반적으로 사용되는 바이오틴 또는 형광 태그와 같은 자신의 기능 일 수 있고, 이하와 생체 활성 (11)을 손상하는 것이 어려울 수 있다는 장점을 가지고있는 광 가교 공정과, 이후 상기 작용을 필요 전구체 일 수있다. 이 프로토콜에서, 우리는 diazirine의 광 가교기를 함유하는 photoaffinity 프로브, 및 구리 (I)을 통해 리포터 그룹의 부착 말단 알킨 -catalyz을 사용한에드 아 지드 - 알킨 샤프 - Hüisgen의 사이클로 (또는 클릭) 반응 12-15. 은 SAR 연구, 설계 및 합성을 조사하고, 이러한 연구의 결과는 다른 곳에서 16-18 발표되었다.

<table><tbody><tr><td>Tris(2-carboxyethyl)phosphine (TCEP)</td><td>Life Technologies</td><td>20490</td><td>Make fresh day of use. Prepare 100 mM stock in water with 4 eq NaOH.</td></tr><tr><td>Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA)</td><td>AnaSpec</td><td>63360-50&amp;nbsp;</td><td>Prepare 1.7 mM stock in a 4:1 ratio of t-butanol to DMSO, store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Copper Sulfate (CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;5H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>LabChem, Inc.</td><td>LC13440-1</td><td>Prepare 50 mM stock in water, store at room temperature.</td></tr><tr><td>Biotin-azide</td><td>Click Chemistry Tools</td><td>AZ104-100</td><td>Prepare 10 mM stock in DMSO, store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Alexa Fluor 647-azide&amp;nbsp;</td><td>Life Technologies</td><td>A10277</td><td>Prepare 1 mM stock in DMSO, store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>365 nm UV lamp</td><td>Spectroline</td><td>FC100</td><td>UV-blocking glasses should be worn while operating.</td></tr><tr><td>Protease inhibitor tablets, EDTA-free</td><td>Roche Life Science</td><td>11873580001</td><td>Prepare 50x solution in water and store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Sonicator</td><td>Branson</td><td>Sonifier 250</td><td>Set to output 1, duty 30%.&amp;nbsp;</td></tr><tr><td>Fluorescent gel scanner</td><td>GE Healthcare Life Sciences</td><td>FLA 9500</td><td>Use red laser to detect Alexa-fluor 647.</td></tr><tr><td>Detergent-compatible Dc protein assay kit</td><td>Bio-Rad</td><td>5000112</td><td></td></tr><tr><td>High Capacity Streptavidin Agarose beads&amp;nbsp;</td><td>Life Technologies</td><td>20359</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagles Medium, low glucose</td><td>ThermoFisher Scientific</td><td>11885092</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified</td><td>ThermoFisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Penicillin/Streptomycin solution</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>SDS Sample Buffer (2x)</td><td>ThermoFisher Scientific</td><td>LC2676</td><td></td></tr></tbody></table>

identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

여기에 표시된 결과는 이전에 16 게시 된 사용하는의 항진균제 이트라코나졸의 사진 친 화성 프로브를 얻었다. 이러한 결과는 성공적 35 kDa의 막 단백질 전압 가변 음이온 채널 1 (VDAC1)와 같은 주요 이트라코나졸 결합 단백질을 확인하기 위해 살아있는 세포 photoaffinity 라벨링 기술의 사용을 입증한다. 상기 프로토콜은 ...

Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

라이브 셀 Photoaffinity 라벨에 의해 기본 셀룰러 환경에서 작은 분자 결합 단백질의 식별

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

identification-small-molecule-binding-proteins-native-cellular

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

12.5K 조회수.  Johns Hopkins University School of Medicine. We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis. 

비디오: Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

Proximity Ligation Assay를 이용한 현탁 세포 배양에서의 DNA 손상 유발 단백질 복합체의 검출 및 가시화

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

연구

생화학

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 ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

사진-가교 매핑 및 라이브 포유류 세포에서 Bioorthogonal 화학 GPCRs에 프로브 화학의 유전 결합을 최적화

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

화학

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

단일 분자 형광 현미경에 의한 수용체 역학의 고해상도 시공간 분석

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

생체공학

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Photoactivated 단일 분자 추적을 사용하여 라이브 박테리아 세포에있는 단백질-DNA 상호 작용을 시각화

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

면역학 및 감염병학

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

이분자 형광 보완과 광 활성화 현지화 현미경 (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

TCR-pMHC 바인딩 측정<em&gt; 현장에서</em&gt; FRET 기반의 현미경 분석을 사용하여

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

COS - 7 형광 라벨링는 라이브 셀 이미징에 대한 SNAP - 태그 퓨전 단백질 표현

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

생물학

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

살아있는 포유류 세포의 세포에서 단일 막 단백질의 다 색 지역화 현미경

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

단백질 상호 작용의 Live Cell Single-Molecule Imaging-Based 정량화

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

저자 스포트라이트: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

생체 분자 응축물 내 단백질 상호 작용 역학의 단일 분자 측정

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...