이 작품은 NIH 보조금 R01NS046835과 R01AG034389, 그리고 천 부여 6FY08 - 322 3 월 지원했다. 특별한 당신이 맥을 기술에 그녀의 통찰력과 함께 엄청난 도움을 부인 엘리 노어 브라운 (Miltenyi Biotec, 오번, CA)에 전념합니다 감사합니다. Miltenyi은 아낌없이 무료로 실험의 시연에 사용되는 자료를 제공하고 있습니다. 또 세포 라인을 표현 PKCζ을 생성 박사 ​​Guanghu 왕 (조지아 / 조지아 보건 과학 대학, 오거 스타의 메디컬 칼리지, GA)에 감사드립니다. 조지아 / 조지아 보건 과학 대학 (박사 린 메이의 지도자의 임기 미만)의 의료 대학에서 분자 의학 연구소에 의해 지원도 인정받고 있습니다.

1. 소개 자기 - 활성화된 셀 정렬 (LIMACS) 기법을 사용하여 지질 소포 - 중재 친화 크로마 토그래피는 ceramide - 관련 단백질 단지가 1-3 분리하기 위해 연구실에서 개발되었습니다. 원래, 지질 vesicles는 vesicles과 관련된 단백질을 분리하는 자기 입자 - 복합 Annexin V를 (phosphatidylserine 고도로 affine)를 사용하여 Mac 용 허용 ceramide와 phosphatidylserine의되었다. 이 ceramide - 연결된 극성 복잡하고 세포에서 ceramide 결합 단백질의 분리의 체외의 reconstitution에 3 lysates 위해 우리는 LIMACS 기술을 사용해 왔습니다. LIMACS은 vesicles (예 : glycolipid - specifc lectins 또는 지질 항체)의 절연 다른 상호 작용 파트너를 사용하여 수정할 수 있습니다. 2. 실험 절차 지질 Vesicles 및 aPKCBinding Assays의 준비 <ol><li> 지질 vesicles은 phosphatidylserine (105 μg)과 대형 리포좀 준비 1,4-7에 대한 변경 절차는 다음과 C16 - ceramide (85 μg)의 몰 금액의 건조 혼합물에서 얻을 수 있습니다. </li><li> 지질 혼합물은 다음 resuspended 50 MM 트리스 / HCL (산도 7.5) 150 MM NaCl로 구성된 소포 버퍼 100 μl 1 H에 대한 sonicated 수 있습니다. </li><li> 0.1 MM MnCl 2 보충 소포 버퍼 300 μl를 추가 후, 샘플 4 20 분 12,000 μ의 g ° C.에 centrifuged 아르 </li><li> 펠렛은 (대형 지질 vesicles) 소포 버퍼 100 μl에 resuspended 37 1 H에 대한 Vybrant CM - diI ​​1 nmol와 incubated 있습니다 ° C가 맥 분리 후 소포 분획을 시각화합니다. Vybrant CM - diI ​​특별히 지질 세포막에 통합 빨간색 형광 염료이다. </li><li> 세제없는 세포 lysate은 원심 분리에 의해 막의 파편의 제거 다음 hypotonic 버퍼 (프로 테아제와 인산 가수 분해 효소 억제제 10 MM 트리스 / HCL (산도 7.0)) 300 μl에 sonication / 세포의 균질로 준비가되어 있습니다. 1 H 100,000 XG에서 원심 분리 단계는 phosphatidylserine를 포함하는 내생 세포막과 세포 lysate의 오염을 방지하기 위해 추가되어야합니다. </li><li> 삭제 lysate은 지질 소포의 정지에 추가하고, 혼합물은 4 ° C. 2 H에 대한 incubated입니다 </li><li> 반응 혼합물은 20x Annexin V 구속력 버퍼와 Annexin V에 복합 자기 구슬을 포함하는 솔루션을 50 μl 20 μl로 보충됩니다 4 1 H ° C.위한 인큐베이션 다음 </li><li> 맥스는 제조 업체 (Miltenyi Biotec (주)) 프로토콜에 따라 수행됩니다. 지질 vesicles의 존재와 수량은 microplate 형광 판독기를 사용하여 흐름 통해 용출 분수에 Vybrant CM - diI의 형광을 모니터링에 의해 결정됩니다. </li><li> 기공을 갖는 지질의 금액과 형광 강도 사이의 선형 상관 관계 소포 - 바운드 Vybrant CM - diI는 Annexin V - 기반 Mac에 적용된 지질 혼합물의 양적 고성능 박막 크로마 토그래피 (HPTLC)에 의해 확인됩니다. </li><li> ceramide / phosphatidylserine vesicles에 aPKC 또는 다른 단백질의 바인딩 반응의 특이성은 ° C 부화하기 전에 4 개의 1 H에 대한 세포 lysate를 부화 방지 PKCζ 토끼 polyclonal 항체의 1 μg을 사용하여 항체의 경쟁 분석에 의해 확인 지질은 vesicles. </li><li> 맥스 eluate에서 ceramide / phosphatidylserine vesicles에 바인딩 단백질은 SDS - PAGE와 immunoblotting으로 분석됩니다. </li></ol> 체외 지질 - 단백질 복합 극성에 <ol><li> 지질 - 단백질 극성 복합 체외의 reconstitution의는 이전 섹션에서 설명한대로 LIMACS 과정을 수행합니다. 간단한, phosphatidylserine (420 μg)과 C16 - ceramide (107 μg)에서 유기 용매의 건조이다. </li><li> 말린 lipids은 소포 버퍼 500 μl (; 150 MM NaCl 50 MM 트리스 / HCL, 산도 7.5)에서 sonication 아래 resuspended 있습니다. </li><li> 10 MM MnCl2, Vybrant CM - diI ​​1 μl, 그리고 PKCζ 500 NG (인간 재조합)의 다섯 μl가 추가되고 반응 혼합물은 4 60 분 undergentle의 교반을 incubated 있습니다 ° C. </li><li> Vybrant CM - diI ​​묻은 phosphatidylserine / ceramide vesicles 4에서 60 분 ° C.에 대한 12,000 XG에서 원심 분리하여 회수 아르 </li><li> 펠렛 (분홍색)을 100 μl 트리스 버퍼에 resuspended 및 γS - GTP (100 μm의), GDP (1 ㎜), GST - Par6 (100 NG) 또는 GST - Cdc42 (500 NG)과 보완 및 추가 3 incubated입니다 4 H ° C (관심 재조합 단백질의 다른 조합이 여기에 사용할 수 있습니다.) </li><li> Annexin V - 버퍼 (20x 재고 솔루션 5 μl)와 Annexin V - 복합 자석 구슬이 (50 μl) 추가하고 반응 혼합물은 4에서 다른 30 ​​분 ° C.에 대한 부드러운 교반하에 incubated 아르 </li><li> Annexin V - 맥스는 이전에 설명한대로 공급 업체의 프로토콜을 다음과 수행됩니다. </li&gt; <li> 용출 분획 (1 ML)는 강수량의 도움으로 순수 ovalbumin 10 μg로 보충합니다. 단백질은 위셀 - 플러그의 석출에 의해 집중하고 이전 8 설명된대로 SDS-PAGE/immunoblotting 의해 분석됩니다. </li><li> eluted 지질 vesicles의 금액은 위셀 플러그의 침전 반응의 유기적 (클로로포름 / 메탄올) 단계에서 Vybrant CM - diI ​​(분홍색)의 검출에 의해 계량입니다. 분석 단백질의 양은 지질 vesicles 동등한 양의에 정상화됩니다. </li></ol> 3. 검색 결과 LIMACS의 PKCζ - EGFP의 정화와 ceramide 바인딩 도메인 C20ζ - EGFP 전체 길이를 표현 MDCK 세포의 세제 무료 lysate PKCζ C - 말기 녹색 형광 단백질 (FLζ - EGFP) 또는 PKCζ (C20ζ - EGFP)의 C ​​- 말단에서 ceramide 바인딩 도메인에 연결된 것은 같은 phosphatidylserine / ceramide vesicles와 incubated되었습니다 실험 절차에 설명되어 있습니다. 맥에서 열 용출 후, 단백질은 immunoblotting 및 eluted 단백질 2의 검출에 대한 PKCζ와 EGFP에 대한 항체를 사용하여 분석했다. <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="그림 1" /> 그림 1. LIMACS EGFP - 라벨 PKCζ과 C - 말단 조각 C20ζ phosphatidylserine / ceramide vesicles를 사용합니다. 실험 절차 섹션에 설명된대로 EGFP (구속력 제어 등), 전체 길이 PKCζ - EGFP 또는 ceramide 바인딩, C - 말단 조각 C20ζ - EGFP을 표현 MDCK 세포의 세제 무료 lysates은 phosphatidylserine / ceramide의 vesicles와 incubated되었습니다 . LIMACS을 사용한 후, 단백질은 SDS 샘플 버퍼 eluted되었으며 SDS - PAGE로 분석 및 immunoblotting. 왼쪽 패널은 EGFP는 Annexin V - 연결된 자석 구슬로 유지 vesicles에 바인딩 않았 것으로 나타났습니다. 중간과 오른쪽 패널은 전체 길이 PKCζ - EGFP와 C20ζ - EGFP 인해 ceramide에 바인딩 유지했다. 보여줍니다

<ol>
	<li>Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+to+ceramide+activates+protein+kinase+Czeta+before+the+formation+of+a+pro-apoptotic+complex+with+PAR-4+in+differentiating+stem+cells.">Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.</a> J Biol Chem. 280, 26415-26424 (2005).</li><li>Wang, G., Krishnamurthy, K., Umapathy, N. S., Verin, A. D., Bieberich, 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+carboxyl-terminal+domain+of+atypical+protein+kinase+Czeta+binds+to+ceramide+and+regulates+junction+formation+in+epithelial+cells.">The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.</a> J Biol Chem. 284, 14469-14475 (2008).</li><li>Chalfant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+ceramide+regulates+the+alternative+splicing+of+caspase+9+and+Bcl-x+in+A549+lung+adenocarcinoma+cells.+Dependence+on+protein+phosphatase-1.">De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.</a> J Biol Chem. 277, 12587-12595 (2002).</li><li>Simon, C. G., Holloway, P. W., Gear, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+C(16)-ceramide+between+phospholipid+vesicles.">Exchange of C(16)-ceramide between phospholipid vesicles.</a> Biochemistry. 38, 14676-14682 (1999).</li><li>Goni, F. M., Contreras, F. X., Montes, L. R., Sot, J., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+(and+sociology)+of+ceramides.">Biophysics (and sociology) of ceramides.</a> Biochem Soc Symp. , 177-188 (2005).</li><li>Kumagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CERT+mediates+intermembrane+transfer+of+various+molecular+species+of+ceramides.">CERT mediates intermembrane transfer of various molecular species of ceramides.</a> J Biol Chem. 280, 6488-6495 (2005).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Anal Biochem. 138, 141-143 (1984).</li></ol>

관심 없음 충돌 선언하지 않습니다.

지질과 결합 단백질 간의 특정 상호 작용을 테스트하려면 것은 세포막의 lipids의 포함에 의해 방해합니다. 세포막은 여러 lipids과 단백질의 혼합물로 구성되어 있으며, 그것은 지질 microdomains이나 보트로 구성됩니다. 단백질 직접 지질에 바인딩 또는 유일한 microdomain 구조에 풍부한 경우 따라서 microdomains 및 단백질의 공동 정화 명확하게 구별 수 없습니다. 이러한 지질 ELISAs 또는 Plasmon 공명 분광법으로 표면에 코팅 정의 lipids를 사용하는 다른 방법은 바인딩 단백질과 특정 지질의 상호 작용을 감지할 수 .. 그러나, 생리학 관련 멤브레인 환경에서 표면 (예를 들어, Plasmon 공명 분광법을위한) 지질 vesicles 또는 liposomes로 코팅되어야한다를이 상호 작용을 결정합니다. 우리는이 이전 방법의 대안으로 소설 지질 소포 바인딩 분석을 개발했습니다. 참신는 Annexin V - 복합 자석 구슬과 지질 vesicles의 절연을 허용 vesicles로 앵커 지질 (phosphatidylserine)를 통합에서 비롯됩니다. 따라서, 우리는 자기 활성 셀 정렬 (LIMACS)를 사용하여이 분석 지질 소포 - 중재 선호도 크로마 토그래피를 칭했다. LIMACS가 내생 단백질에 대해 수행할 수, 세포, 또는 재조합 단백질의 표현 단백질은 지질 단백질 복합에 1-3 재구성. 지질 vesicles에 바인딩 단백질 그런 다음 단순히 자석 스탠드에서 맥 컬럼을 복용하고 적절한 버퍼로 eluting하여 복구할 수 있습니다. 이것은 SDS - PAGE를 사용하고 immunoblotting 단백질 분석을 수행할 수있는 eluate 또는 SDS 샘플 버퍼에있는 효소 활동을 측정하는 효소 호환 버퍼 수 있습니다. SDS 샘플 버퍼를 사용하는 경우 맥을 열은 스탠드에 자석 구슬을 보유를 허용 마그네틱 스탠드에서 제거 필요가 없습니다. LIMACS를 사용할 때주의가 필요하다 예방 조치가 있습니다. 이것은 지질 vesicles를 파괴하기 때문에 결국, LIMACS는 세제 lysate와 함께 사용할 수 없습니다. 또한이 지질은 Annexin V - 복합 자석 구슬에 vesicles의 바인딩을위한 앵커로 사용하기 때문에 대상 지질의 바인딩 단백질도 phosphatidylserine에 바인딩하는 경우 테스트되어야한다. . 어떤 경우에는, phosphatidylserine의 타겟 단백질에 바인딩을 해칠 수 있습니다. 따라서, phosphatidylserine의 다양한 양의와 부정적인 컨트롤과 컨트롤은 분석에 포함해야합니다. Thesecontrols 쉽게에만 phosphatidylserine 또는 구속력 lipids과 phosphatidylserine의 혼합물을 포함하는 지질 vesicles를 사용하여 수행할 수 있습니다. 포함 부정적인 컨트롤도 내생 phosphatidylserine의 상당한 금액을 포함하는 세포 lysates 필요합니다. 그것이 1 H. 100,000 XG에서 ultracentrifugation 단계를 포함하여 세포 lysate을 취소하는 것이 좋습니다 내생 지질 세포막이나 vesicles과 오염을 방지하려면 그것은 표면에 뜨는에서 지질 바인딩 단백질에만 LIMACS 절차의 제한 사항입니다 cytosolic, 수있다 분명하다. 이러한 GM1과 자석 구슬과 함께 복합 콜레라 독소 B 급 subunit 같은 다른 앵커 lipids에 LIMACS을 확장할 수있는 가능성이있다. 우리는 현재 vesicles의 준비를위한 앵커 lipids의 사용이 필요 할거 LIMACS에 대한 지질 특정 항체의 사용을 모색하고 있습니다. LIMACS의 유익한 측면 중 하나는 implementable되지 않은 단백질 분석 방법의 다양한 수있는 지질 단백질 복합의 격리는 ... 예를 들어, 우리는 ceramide - 바운드 aPKC과 관련된 Par6/Cdc42의 재구성 극성 단백질 복잡한 분리 LIMACS를 사용했습니다. 따라서, LIMACS는 지질 관련 단백질 단지의 분석 및 분리를위한 강력한 새로운 방식입니다.

Annexin V - 복합 자성 구슬과 맥을 마이크로와 minicolumns은 Miltenyi Biotec, 주식 회사 (오번, CA)에 의해 제공되었다. 모든 lipids은 최고 순도되었으며 아반티 폴라 Lipids (엘라 베스터, AL)에서 얻은.

lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction

lipids 대부분의 정화 절차에 접근할 그러므로 세포 점막에 포함된하고 있기 때문에 지질 단백질 상호 작용의 분석 어렵습니다. 대안으로, lipids은 지질 엘리사와 Plasmon 공명 분광법을 위해 사용되는 평면 표면에 코팅하실 수 있습니다. 그러나, 표면 코팅 lipids은 지질 바인딩 속성에 대해 중요한 수있는, microdomain 구조를 형성하지 않습니다. 또한, 이러한 방법들은 목표 lipids에 바인딩 단백질의 대용량의 정화를 위해 허용하지 않습니다. 지질 단백질 상호 작용을 테스트 이러한 한계를 극복하고 지질 바인딩 단백질을 정화하기 위해 우리는 자기가 활성화된 셀 정렬 (LIMACS)를 사용하여 지질 소포 - 중재 친화 크로마 토그래피를 칭했다 소설 방법을 개발했습니다. 이 방법에서는 지질 vesicles는 Annexin V Mac 용 앵커 지질로 대상 지질 및 phosphatidylserine와 준비가되어 있습니다. Phosphatidylserine는 단백질 Annexin V. phosphatidylserine 함유 지질 vesicles는 자성 비즈에 바인딩됩니다 Annexin V에 복합 자기 구슬을 사용하여 높은 친화력을 보여주는 유비 쿼터스 세포막의 인지질이다. 지질 vesicles이 lysate 세포와 incubated 경우 대상 지질에 바인딩 단백질도 비즈에 바인딩되며, 맥을 사용하는 공동 정화 수 있습니다. 이 방법은 테스트하는 데 사용할 수있는 재조합 단백질은 reconstitute 대상 지질에 바인딩 단백질 컴플렉스를하는 경우. 우리는 sphingolipid ceramide와 공동 정화 전립선 apoptosis 반응 4 (파 4), ceramide - 관련 aPKC에 바인딩 단백질 비정형 PKC (aPKC)의 상호 작용을 표시하기 위해이 방법을 사용했습니다. 우리는 또한 세포 극성과 관련된 단백질 Par6 및 Cdc42와 재조합 aPKC의 ceramide - 연결된 복합 reconstitution이 방법을 사용했습니다. 지질 vesicles가 sphingo 또는 phospholipids의 다양한 준비할 수 있기 때문에 LIMACS은 세포막의 밀접하게 유사한 지질 환경에서 지질 - 단백질 상호 작용에 대한 다양한 테스트를 제공합니다. 추가 지질 단백질 단지는 지질 바인딩 단백질 지질 vesicles 공동 정화의​​ proteomics 분석을 사용하여 확인할 수 있습니다.

Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

우리가 맥스와 Annexin V - 복합 자석 구슬과 대상 지질 및 Annexin V - 구속력 phosphatidylserine의 합성 지질 vesicles를 사용하여 자사의 목표 지질과 단백질의 상호 작용을 테스트하려면. 대상 지질에 바인딩 단백질은 공동 정화 및 비즈에서 용출 후 분석하고 있습니다.

자기 활성 셀 정렬 (LIMACS)를 사용하여 지질 소포 - 중재 동질 크로마 토그래피 : 단백질 지질 상호 작용을 분석하는 소설 방법

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

lipid-vesicle-mediated-affinity-chromatography-using-magnetic

Nanyang Technological University

Research

JoVE Journal

Biology

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

12.4K 조회수.  Georgia Health Sciences University. 우리가 맥스와 Annexin V - 복합 자석 구슬과 대상 지질 및 Annexin V - 구속력 phosphatidylserine의 합성 지질 vesicles를 사용하여 자사의 목표 지질과 단백질의 상호 작용을 테스트하려면. 대상 지질에 바인딩 단백질은 공동 정화 및 비즈에서 용출 후 분석하고 있습니다.

비디오: Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

식물 Protoplasts의 형광 활성 세포 정렬

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

연구

생물학

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

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

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

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

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 ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

헬리 우스 진 건을 사용하여 양파 세포에서 단백질 - 단백질 상호 작용에 대한 Bimolecular 형광 Complementation (BiFC) 어세이

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (AVEXIS)

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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 ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

의 세포 구체적인 분석<em&gt; Arabidopsis</em&gt; 형광 활성화 셀 정렬을 사용하여 잎

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

셀 추적 염료를 사용하여 전지 부문 모니터링에 최적화 된 착색 및 확산 모델링 방법

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

어머니 세포 및 변동 시험의 자기 정렬을 결합에서 유사 분열 세포 노화 동안 게놈 불안정성을 분석합니다<em&gt; 사카로 미세스 세 레비 시아</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

탠덤 선호도 정화를 사용하여 MyoD 상호 작용 체의 식별 질량 분석에 결합

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

마그네틱 활성화 전략을 분리 하 고 정화 활 액 액체 파생 된 중간 엽 줄기 세포는 토끼 모델에서 정렬 셀

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...