이 작품은 VF에 국립 보건원 (NS42599 및 GM077569)에서 보조금에 의해 지원되었다.

1. Crosslinking 준비 <ol><li> 당신은 표준 튜브 반응 당 단백질 500 μg의 고립을 허용 접시에 세포의 충분한 번호가 필요합니다. 하나의 튜브가 immunoblot으로 관심의 단백질 putative interactors의 식별하기에 충분한 수 있습니다. 이 경우 비드 바운드 자료는 SDS - PAGE 샘플 버퍼 (immunoprecipitation)로 eluted 수 있습니다. 질량 분광법의 분석을 위해, 표준 반응의 숫자는 적어도 10 배 증가되어야하며 단백질 단지는 (면역 친 화성 크로마 토그래피) 사용 항체에 의해 인식 펩타이드 항원과 함께 밖으로 경쟁에 의해 eluted해야합니다. 이 전략은 면역 글로불린 무료 단백질 단지의 격리를 허용합니다. </li><li> crosslinking에 대한 최적의 세포 밀도가 75 % confluency 90% 사이입니다. 높은 confluency 조건에서, 세포는 모든 세포에 세포 투과 crosslinker에 접근을 감소하는, 서로에 대한 더미하는 경향이있다. </li><li> 세포 종류와 실험 조건은 셀 플레이트의 하단에 표시해야합니다. 우리는 일상적으로 혼자 자동차와 컨트롤을 포함합니다. </li><li> 이전 crosslinking에 DSP 솔루션을 제외한 모든 솔루션을 준비합니다. <ol><li> CaCl 2 0.1 MM 및 CaCl 2 실험을 시작하기 전에 1 ㎜ (PBS / CA / MG)과 인산염 버퍼 호수 버퍼의 재고를 준비합니다. 칼슘 및 마그네슘 이온의 또한이 실험의 과정 동안 문화 판에 세포 접착을 위해 중요합니다. 4 스토어 ° C. </li><li> 50X 완료 테아제 억제제 칵테일 - 1mL 밀리 Q의 물속에 녹아있는 한 타블렛. -20 ° C.에 저장 </li><li> 20% 트리톤 X - 100은 10g 트리톤 Triton X -100 밖으로 체중 50 ML 밀리 Q 워터의 전체 볼륨에 희석. 4 록 ° C 4 박 및 저장 ° C까지 사용. 이상 1 개월 보관하지 마십시오. 아래에 설명된 용해 및 IP 버퍼를 준비하는이 20 % 주식 dilutions을 사용합니다. </li><li> 산도 7.4에 50X DSP 칭 솔루션 1M 트리스. 상온에서 보관하십시오. </li><li> 10X 버퍼에게 솔루션을 준비합니다. 10X 버퍼 : <ul><li> 50 ML 1 M HEPES </li><li> 150 ML 5 M NaCl </li><li> 10 ML 0.5 M EGTA </li><li> 250 μL 2 M MgCl 2 </li><li> 7.4으로 산도를 조정 </li><li> 500 ML에 마지막으로 볼륨을 가지고 </li></ul> 10X 버퍼 솔루션은 필요에 따라 1X 버퍼로 희석합니다. 1X 버퍼는 또한 lysate와 단백질 자석 - 강수량 버퍼에 사용됩니다. 1X 버퍼 <ul><li> 10 MM Hepes </li><li> 150 MM NaCl </li><li> 1 ㎜ EGTA </li><li> 0.1 MM MgCl 2 </li><li> 산도 7.4 </li></ul></li><li> 용해 버퍼 1X 버퍼 + 0.5 % 트리톤 X - 100. </li><li> 단백질 마그네틱 강수량의 버퍼 (IP 버퍼) 1X 버퍼 + 0.1 % 트리톤 X - 100. </li></ol></li></ol> 2. Crosslinking 솔루션 준비 <ol><li> 즉시 세포에 적용하기 전에 DSP 솔루션을 준비합니다. DSP는 매우 소수성이며 PBS / CA / MG 버퍼로 diluting 전에 DMSO에 용해한다. DMSO 1 ML에 DSP의 40 밀리그램을 디졸브. 이것은 DSP (DSP의 분자량은 404.42 g / 몰입니다)의 100 MM 솔루션을합니다. </li><li> PBS로 DSP / DMSO의 희석을 용이하게하기 위해서 ° C에서 37로 PBS / CA / MG의 적절한 볼륨을 따뜻하게. 여러 플레이트 크기에 필요한 볼륨 : 물론 당 6 - 음 판 2 ML 판 당 10cm 플레이트 10 ML 판 당 15cm 플레이트 20 ML </li><li> 따뜻한 PBS / CA / MG의 모든 1 ML위한 DSP / DMSO 재고 솔루션의 10μL를 추가합니다. 모든 DSP가 해산 때까지 반복 혼합과 드롭하여 DSP / DMSO 재고 솔루션 드롭을 추가합니다. PBS / CA / MG의 모든 1 ML에 추가 10 μL DMSO의 제어 솔루션을 확인하십시오. </li><li> 10 분 이상 더 이상 얼음 물을 욕조에있는 모든 PBS / CA / MG 솔루션을 놓으십시오. </li></ol> 3. Crosslinking에 대한 셀 준비 <ol><li> crosslinking 또는 차량 제어 보육에 대한 모든 번호판을 맞게 얼음 물 목욕을 준비합니다. </li><li> 37 ° C 배양기에서 접시를 타고 얼음 물을 욕조에 바로 놓으십시오. </li><li> 얼음처럼 차가운 PBS / CA / MG와 세포 두 번 씻으십시오. 당신은 (섹션 2.2 참조) crosslinker 배양에 사용되는 같은 볼륨을 사용합니다. </li><li> 두번째 세척을 제거하고 차량 제어 또는 DSP의 crosslinking 버퍼 솔루션 중 하나를 추가합니다. </li><li> 두 시간 동안 얼음에 품어. 당신은 접시 모든 세포 솔루션이 적용되는 것을 보장하기 위해 대략 매 20 분 확인하시기 바랍니다. 당신은 DSP의 작은 금액이 솔루션의 밖으로 침전물이 나타날 수 있습니다. 이것은 정상입니다. </li></ol> 4. DSP 반응의 불활 성화 <ol><li> PBS / CA / MG (PBS / CA / MG의 모든 1 ML 1 M 트리스 산도 7.4의 20 μL) 20 MM 트리스 산도 7.4의 1X DSP 담금질 솔루션을 준비합니다. </li><li> 차량 제어 및 crosslinking 솔루션을 제거합니다. </li><li> 얼음처럼 차가운 불활 성화 솔루션을 추가하고 15 분 동안 얼음에 품어. </li></ol> 5. 세포 용해 <ol><li> DSP 담금질 보육 동안 0.5 % 트리톤 버퍼에 X - 100과 함께 완전한 테아제 억제제 칵테일의 세포 용해 버퍼를 준비합니다. 모든 1 ML의 용해 버퍼에 대한 완벽한 20 μL 50X 재고 솔루션을 추가합니다. </li><li> 십오분 DSP 담금질 보육 다음, PBS / CA / MG와 세포 두 번 씻는다. </li><li> 30 분 용해 버퍼 + 4 셀 및 바위 완료 ° C를 추가합니다. 일부는 여러 플레이트 크기에 대한 용해 버퍼의 볼륨을 제안 : <ul><li> 물론 당 6 - 잘 플레이트 0.5 ML </li><li> 판 당 10cm 플레이트 1 ML </li><li> 판 당 15cm 플레이트 1 ML </li></ul></li><li> 용해 후, 접시에서 세포를 수집하는 세포 스크레이퍼를 사용합니다. 단백 분해 효소 활동을 최소화하기 위해 얼음에 lysates을 유지하시기 바랍니다 </li><li> 스핀 셀룰라 최고 속도 (15.000 XG)에서 벤치 탑 미니 원심 분리기 4 15 분 ° C에 대한 lysates. </li><li> 새로운 튜브에 뜨는를 피펫. 펠렛 폐기하십시오. </li><li> 단백질 수준을 분석하고 immunoprecipitation과 함께 진행합니다. </li></ol> 6. 단백질 자기 강수량 비즈 준비 참고 :이 단계는 일반적으로 직접 2 시간 crosslinking 보육을 시작 후에 시작됩니다. <ol><li> 30 Dynal immunomagnetic 구슬 μL, IP 버퍼 500 μL (1X 버퍼 + 0.1 % 트리톤 X - 100) 및 microcentrifuge 튜브를 screwtop하는 항원 특정 항체의 해당 금액을 결합합니다. 부정적인 컨트롤에 대한 항체가없는가 아닌 특정 항체 튜브 준비합니다. 항체의 적절한 금액은 별도의 실험을하는 동안 각각의 항원에 따라 결정되어야합니다. 모든 IP 튜브 라벨. </li><li> IP 튜브가 두 시간 동안 실온에서 엔드 오버 엔드 로커에서 부화 수 있습니다. 또는, crosslinking하기 전에 4 명이 하루 밤새 항체로 IP 튜브를 품어. </li><li> 부화 후, 튜브의 하단에있는 비즈를 수집하는 빠른 10초 미니 원심 분리 해. </li><li> 멀리 튜브의 바닥에서 자기 구슬을 끌 수있는 자석 홀더로 튜브 슬라이드. 안전하게 비켜 비즈와 함께, 당신은 지금 쉽게 어떤 언바운드 항체를 멀리 세척하실 수 있습니다. </li><li> 이러한 겔 로딩 팁 또는 부화 버퍼 및 언바운드 항체를 제거하는 p200 팁 등 작은 구멍 흡인기 팁을 사용합니다. 부화 볼륨이 제거되자 마자 IP 버퍼 1 ML에 추가합니다. </li><li> 부드럽게 다시 중단 비즈, 튜브를 캡 및 엔드 오버 엔드 회전 5 분 실온에서 모든 IP 튜브를 품어. </li><li> 한번 더 IP 버퍼의 새로운 밀리리터로 5 분 세척 단계를 (6.6 단계를 통해 6.3) 반복합니다. </li><li> 가교 lysate의 비즈에 추가 준비가 될 때까지 IP 튜브는 지난 씻어 보육에 남아있을 수 있습니다. </li></ol> 7. 단백질 자성 비즈와 가교 Lysate를 품어 <ol><li> 마지막 세척 회전 단백질 자석 구슬 후, 아래로 10 초 동안 미니 원심 분리기 인치 그런 다음 자석 홀더에 튜브를 살짝로 진행합니다. </li><li> 다시, 작은 구멍 열망 팁을 사용하면, 튜브에서 부화 볼륨을 제거합니다. 즉시 단백질 자석 구슬이 들어있는 튜브에 가교 세포 lysate (1 μg / ML에서 lysate를 가정) 500 μL를 추가합니다. 당신은 부정적인 제어로 펩타이드 경쟁을 사용한다면, 펩타이드의 적절한 금액은이 시점에서 포함되어야합니다. 이전 실험은 적절한 펩타이드의 경쟁 조건을 결정하기 위해 수행되어야합니다. 또는, 당신은 부정적인 제어로 lysate 무료 단백질 자석 구슬을 할 수 있습니다. 이 경우에는 즉시 단백질 자석 구슬에 + 완료 Lysate 버퍼 (1X 버퍼 + 0.5 % 트리톤 X - 100) 500 μL를 추가합니다. </li><li> 부드럽게 구슬을 resuspend. Resuspended 구슬은 2 시간 엔드 오버 엔드 회전에서 4 명이에 incubated해야합니다. </li></ol> 8. 비즈에서 언바운드 Lysate 와시 <ol><li> 일단 lysate는 미니 원심 분리기의 빨​​리 10초 스핀은 충분한 부화 시간을 가지고 있습니다. 그런 다음 튜브는 자석 홀더에 끼웁니다. 자석 홀더는 실험의 나머지 얼음 욕조에 보관해야합니다. </li><li> 작은 구멍 흡인기 팁을 사용하여 모든 언바운드 lysate를 제거합니다. 모든 언바운드 lysate를 제거 직후 것은 IP 버퍼 1 ML를 추가합니다. </li><li> 튜브 뚜껑 부드럽게 구슬을 resuspend. </li><li> 일단 구슬이 비드 pelleting 단계 (8.1)을 반복 resuspended 있습니다. </li><li> 다시 부화 볼륨의 모든을 대기음 즉시 IP 버퍼 1 ML을 추가한 다음 튜브를 마개. </li><li> 부드럽게 IP 버퍼에있는 구슬을 resuspend. 엔드 오버 엔드 락을 함께 4 명이시 5 분 resuspended 구슬을 품어. </li><li> 비드 세척 단계를 (8.6 통해 8.4) 4 번 이상 반복합니다. </li></ol> 9. 샘플 변성과 비즈의 수집 <ol><li> 로 단백질 자석 강수량 튜브의 모든 바뀌어진 후, 다시 침전 비즈 (8.1)와 슬라이드 튜브자석 홀더. </li><li> 단백질 자석 구슬에 바인딩 단백질이 조건을 denaturing에서 제거됩니다. 마지막 IP 버퍼 세척을 기음과 자석 홀더에서 IP 튜브를 제거합니다. </li><li> 또 튜브의 하단에있는 수영장에서 구슬을하는 비드 펠렛 위의 IP 튜브에 1X 젤 샘플 버퍼를 추가합니다. 샘플 버퍼의 양은 사용하는 젤의 잘 크기에 따라 달라집니다. 당신은 부드럽게 젤 샘플 버퍼에 resuspended 구슬을 모두 얻을 수있는 튜브의 바닥을 도청 할 수 있습니다. </li><li> 각 IP 튜브 denaturing 과정 (9.3 통해 9.2)를 반복합니다. </li><li> 젤 샘플 버퍼에 resuspended 단백질 자석 구슬은 다음 75 온수 아르 ° denaturing 프로세스를 완료하기 위해 5 분간 C. </li><li> 일단 예제가 서부 모래 바닥에 대한 SDS - PAGE 젤에서 실행할 수있는 변성되었습니다. 비운 단백질 자성 비즈는 원심 분리하여 다시 pelleted과 자석 홀더와 샘플에서 제거할 수 있습니다. </li></ol> 10. Antigenic의 펩티드와 가교 단지 (면역 친 화성 크로마 토그래피)의 용출. <ol><li> 단백질 자석 immunoprecipitation 튜브의 모든 후 자석 홀더에 다시 침전 비즈 (8.1)와 슬라이드 튜브는 씻어되었습니다. 뜨는을 기음과 버퍼 10 μl를 추가 단백질 단지의 immunoisolation에 사용되는 항체에 의해 인식 antigenic의 펩타이드와 함께 보충. 당신이 효율적인 용출에 대한 10-200 μm의까지 농도를 테스트해야합니다. </li><li> 4 2 H에 대한 구슬을 품어 ° C. </li><li> 자기 스탠드에 구슬을 넣어 조심스럽게 eluted 소재의 10 ML을 복구합니다. 이 표면에 뜨는를 저장합니다. </li><li> 신속 버퍼와 비즈를 세척하고 세척을 삭제. 구슬을 저장합니다. </li><li> 1X의 농도에 저장된 뜨는와 구슬로 젤 샘플 버퍼를 추가합니다. ° C 5 분 75 다음 튜브를 품어. </li><li> SDS - PAGE에 의해 구슬과 펩타이드 eluate를 분석합니다. eluate 모두 SDS - PAGE의 얼룩 단백질이나 immunoblots (그림 1B)에 의해 IgG 무료로해야합니다. IgG 무료이 자료는 질량 분광법에 적합합니다. </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="그림 1" /> 그림 1. AP - 3 상호 작용하는 단백질 합성물과 막 단백질의 분리. HEK293 세포 (A) 또는 PC12 세포 (B) 차량 제어의 존재에 중 처리 (DMSO, 홀수 차선 그림 1A) 또는되었습니다 DSP (심지어는 골목길이 그림. 1A 또는 모든 그림의 1B에서 차선). 그림, 명확히 추출물 혼자 비즈 (그림 1A, 레인스 3-4), 트랜스페린 수용체 항체 (그림 1A, 레인스 5-6) 또는 AP - 3 δ 항체 (그림 1A, 레인스 70-10과 중 incubated되었습니다 . 1B, 골목길 10-10). 면역 단지는 SDS - PAGE 샘플 버퍼 (그림 1A)와 eluted했고 혼자 버퍼 또는 (그림의 1B, 차선 1-2), 또는 아미노산 680 710에 해당하는 δ의 antigenic의 펩타이드의 증가 농도와 보충 버퍼 (그림의 1B, 차선 3-10) :; 인간 δ - adaptin (1,923,266 GI AAD03777 NCBI)의. 이 펩티드는 δ 항체를 바인딩합니다. 펩타이드 용출 후, supernatants (S)과 구슬 (B)가 immunoblot (그림의 1B)에 의해 분석되었다. ;뿐만 아니라 clathrin 중쇄 (CHC), 공산권 - 1 subunit의 pallidin, 그리고 홉스 복잡한 subunit의 vps33b : δ, β3 및 σ3 subunits에 대한 항체로 검색된 AP - 3, 다음과 같은 수용성 요소로 coprecipitates 멤브레인는 phosphatidylinositol - 4 - 키나제 타입 II 알파 (PI4KIIα)와 아연 전송 3 (ZnT3)을 단백질. 펩타이드의 IgG 마우스 무거운 체인의 부재는 그림 뜨는을 eluted합니다. 1B.

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP, 멤브레인 - 침투성, 12의 스페이서 암과 화학적 줄이할 수있는 crosslinker는 과도 단백질 상호 작용 1,2,3,4를 안정화하는 데 사용됩니다. 여기서 우리는 어댑터 복잡한 AP - 3 5 endosomes에서 vesicles에 막 단백질을 인식하고 각종 가용성 단백질 복합이 전략을 exemplified. AP - 3 선택 아연 수송 ZnT3 및 지질 키나제 phosphatidylinositol - 4 - 키나제 타입 II 알파지만 트랜스페린 수용체 1,4,6에</...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>		</tbody>
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isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

세포 기계의 동적 특성은 자주 일시적 및 / 또는 약한 단백질 연결에 내장되어 있습니다. 이러한 낮은 친화도 상호 작용 관심의 단백질 주변의 분리 및 단백질 네트워크의 식별에 대한 엄격한 방법을 배제. 화학 crosslinkers의 사용은 따라서 정화에 대한 생화 학적 한계를 거치지 않고 불안 정한 상호 작용의 선택 안정화 수 있습니다. 여기에 우리가의 스페이서 팔로 homobifunctional crosslinker를 사용하는 문화의 세포에 대한 의무 프로토콜을 현재 12, dithiobis - (succinimidyl proprionate) (DSP). DSP는 분자에 존재 disulphide 결합의 감소에 의해 죽습니다. 자석 구슬과 관심의 단백질의 면역 크로마 토그래피와 결합 가교 달리 정화 견딜 않을 단백질 단지의 절연을 수 있습니다. 이 프로토콜은 일반적인 서양 얼룩 기술과 호환되며 그것은 대량 분석법 1 단백질 식별을 위해 확장할 수 있습니다. 스테파니 A. Zlatic와 진주 V. 라이더이 작품에 동일하게 기여.

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

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

세포 투과 crosslinker DSP [dithiobis - (succinimidyl 프로피 온산)] 일시적 안정화와 불안 정한 상호 작용<em&gt; 생체내에</em엄격한 단백질 복잡한 정화 기술을 사용하여 격리 수 있습니다&gt;. 여기 immunoprecipitation에 의해 단백질 단지의 절연 다음 문화의 성장 crosslinking 전지에 대한 기술을 제시한다.

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

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

isolation-labile-multi-protein-complexes-vivo-controlled-cellular

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Biology

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

17.2K 조회수.  Emory University. 세포 투과 crosslinker DSP [dithiobis - (succinimidyl 프로피 온산)] 일시적 안정화와 불안 정한 상호 작용 생체내에. 여기 immunoprecipitation에 의해 단백질 단지의 절연 다음 문화의 성장 crosslinking 전지에 대한 기술을 제시한다.

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

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

사진 유발 가교 수정되지 않은 단백질 (PICUP) Amyloidogenic 펩티드에 적용

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

연구

생물학

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

단일 분자 단지를 떠올리 VIVO에서 고급 형광 현미경을 사용하여

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

진균 독소의 검출을위한 ELIME (효소가 단백질 자기 전기를 연결) 방법

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Macromolecule - 리간드 친화력을 측정하기위한 등온 적정의 열량

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

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 ...

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 ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

셀 벽의 탄성 특성의 AFM 기반의 매핑 : 조직, 세포 및 세포 이하의 해상도에서

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

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) ...