우리는 공촛점 현미경과 관련하여 도움이 박사 스콧 헨더슨을 인정하고 싶습니다. 이 연구는 WAB 현미경으로 의학의 매시 암 센터 및 학교 (VCU) VCU - 도서관에서 공연되었다에서 WAB로 건강 1RO1CA127501 국립 연구소에서 보조금뿐만 아니라 파일럿 프로젝트 기금에 의해 지원되었다. 의 신경 생물학 및 NIHNINDS 센터 핵심 부여 5P30NS047463에서 자금 일부 지원 해부 현미경 시설,,,.

프로토콜은 세 가지 주요 단계로 구성되어 있습니다. 포유류의 표현이 아미노 말기 CFP 및 / 또는 YFP의 monomeric 버전 벡터에만 간략하게 논의한다에 관심을 귀하의 유전자의 첫 번째 단계는 복제. 두 번째 및 세 번째 단계, EA.hy926 내피 세포에 벡터 DNA의 transfection과 초조와 공촛점 이미징은 아래에보다 심도있게 설명되어 있습니다. 1. CFP 및 YFP 키메라 수용체의 건설 관심 수용체는 CFP와 YFP의 monomeric 향상된 버전 아미노 말기 복제해야합니다. 안달의 결정은 경험이고 transmembrane이 지역을 걸치는하고 형광단 1 시작 사이의 링커의 길이에 따라 매우 달라집니다. 따라서, 링커의 여러 가지 길이가 조사를 받고 새로운 각 수용체의 쌍에 대한 탐색해야합니다. <ol><li> monomeric C 또는 YFP (이 벡터는 우리 실험실에서 얻을 수있다)이있는 pcDNA3.1 hygro R 또는 네오 R 벡터로, NheI / EcoRI 사이트에 관심을 귀하의 수용체를 복제. * 참고 - 필요한 경우, NheI가 SpeI 및 XbaI 포함한 호환 여러 효소이다. </li><li> 적절한 endA1 recA 능력 세포주 (우리가 정기적으로 Mach1 사용)로 결합 반응을 변환. 삽입의 존재를 화면 식민지는 DNA가 변이의 부재를 확인하는 순서로 따라갔다. </li><li> 성공적인 분자 복제의 형태를 취득시, Qiagen, 또는 그와 유사한, DNA 키트를 사용하여 소독 물 또는 TE에 transfection 학년 벡터 DNA를 준비합니다. UV 분광법을 사용하여 농도 및 순도를 평가합니다. DNA의 일반적인 수확량이 정기적으로 1 μg / μL의 작업 주식에 희석는 100-200 μg의 범위에 있어야합니다. </li></ol> 2. EA.hy 926 셀의 Transfection <ol><li> MatTek coverslip (제 0) 2 ML 매체로 35mm 요리에 완전한 - DMEM 재배 EA.hy 926 세포 4 (DMEM 10% 태아 소 혈청 + Pen. / Strep.) 분할. 당신은 conservatively 15cm 합류 요리에서 ~ 15 요리 (~ 90 % 합류 다음날)을 얻을 수 있습니다. 각 수용체의 표현뿐만 아니라, 각각의 수용체 쌍을 하나의 요리를 평가 한 접시를 준비합니다. </li><li> 세포 37 5 % CO 2 분위기에서 하룻밤 부화 허용 ° C. 다음날, 문화 ~ 70~90%의 합류해야합니다. </li><li> transfection의 날, 미리 예열 OptiMEM 200 μL에 Fugene6 8 μL와 키메라 수용체 벡터 DNA (2 μL) 2 μg을 혼합하여 nucleolipid 단지를 준비합니다. 2 μg의 총 각 수용체 1 μg과 수용체 쌍 들어, transfect (아래 토론도 참조). </li><li> 역전에 의해 부드럽게 섞는다. </li><li> 30 분 상온에서 부화 계속합니다. </li><li> 잠복기 동안 transfection을 위해 세포를 준비합니다. 신선한 미디어를 대기음 항생제없이 두 ML 사전 예열 완료 - DMEM (DMEM + FBS)의 추가되기 전에 따뜻한 PBS로 부드럽게하고 잠시 각각의 접시를 씻으십시오. 그들이 Fugene6 치료 기간 동안 상당히 많은 독성 세포에 있으므로 페니실린과 스트렙토 마이신의 존재를 방지하는 것이 중요합니다. </li><li> 세포 현명 복잡한 드롭을 추가하고 37 추가 20~24시간에 대한 품어 ° 5 % CO 2 분위기에서 C 있습니다. </li><li> 24 시간 후에 조심스럽게 세포에서 미디어를 대기음 항생제와 함께 신선한 완전한 - DMEM을 피펫. transfection에 따라 세포 생존 능력이 상대적으로 높은 유지한다, 아니 부유 세포 거의가 관찰해야합니다 즉. </li><li> 유전자 발현은 추가 48~60시간 포스트 transfection 후 일반적으로 최대한입니다. 그러나, 영상 24 시간처럼 짧은에서는 일반적으로 가능합니다. 최적의 유전자 발현 시간 코스 실험을 실시하여야한다. </li></ol> 3. 공촛점 이미징 및 분석 걱정 라이브 셀 이미징은 청색 다이오드 (405 nm의), 아르곤 (458, 476, 488, 514 NM)이 장착된 Leica TCS - SP2 AOBS 공촛점 레이저 스캐닝 현미경에서 수행됩니다. 녹색 HeNe (543 NM), 오렌지 HeNe (594 NM), 그리고 붉은 HeNe (633 NM) 레이저, HCX PI APO 63x/1.3 나 글리세린 - 침지 목표 렌즈, 전동 XY 스테이지 (Märzhäuser) 및 환경 제어 ( 온도, 습도 및 CO 2) 단계 인큐베이터 (PeCon). 실험 컨트롤 형광단 방출 채널뿐만 아니라 평가하는 표현의 문제가 아닌 특정 형광단의 multimerization 사이의 교차 대화를 제거하는 중요하다. 따라서, 단일 형광단의 transfections는 모든 이미지 세션을 설정 활용하고 있습니다. 부정의 배경을 결정하기 위하여 수행되어야합니다 컨트롤을 (알려진 noninteracting 수용체를 사용하는) 걱정하는 것은 이상의 표현으로 인해 발생되는 무서워. <ol><li> CFP와 YFP에 대한 방출 채널 간의 누화를 제거하려면 단계 인큐베이터에 CFP 표현 컨트롤을 놓습니다. 하얀 빛을 사용에 초점을 Z - 플레이트를 조정세포 수 있습니다. </li><li> 465-505 NM 및 YFP 525-600 nm의에로 CFP의 방출 감지 SP 창 설정을 설정합니다. </li><li> 모두 방출 채널을 모니터링하는 동안, 458 nm의에서 CFP를 자극. AOTF를 사용하여 YFP 방출 채널로 CFP 이상의 감지할 수있을 정도의 출혈을 제거하기 위해 레이저 전원을 설정합니다. 이 설정을 저장합니다. </li><li> 단 YFP을 표현 세포에 대한 514 nm의 여기에서 전력을 조정하여 CFP 채널에서 YFP 누화 (YFP 표현 컨트롤을 사용)에 대해 동일한 제어를 수행합니다. 이 설정을 저장합니다. </li><li> 다음 장소 세포 공동 표현 무대 인큐베이터에 CFP와 YFP 있습니다. Leica 공촛점 소프트웨어의 순서 설정을 사용하여 적절한 멤브레인 현지화와 CFP와 YFP 비슷한 수준을 표현하는 세포를 찾습니다. </li><li> Leica 소프트웨어 수용체 사진 표백 프로그램을 사용하여, respectably, 저장된 CFP 및 YFP 설정으로 기증자 및 수용체 설정을 설정합니다. </li><li> 세포에 확대, YFP의 사진 파괴는 세포 막에서 발생하고 프로그램을 시작할 수있는 투자 수익 (ROI)을 (관심 영역) 강조. </li><li> 수용체의 사진 파괴 들면, 514 nm의 100 %에 AOTF를 조정하고 YFP 방출의 70 %가 소진되었습니다 때까지 계속 표백 레이저를 허용합니다. 표백 가속, 로아는 일반적으로 레이저의 래스터 축 (X 축)에 선정되었습니다. </li><li> 사진 표백 동안, 이러한 측정치가 false로 효율성을 걱정 보고할 수 있듯이, 세포 운동을 모니터하고 XY - 비행기에서 감지할 수있을 정도의 운동과 함께 얻은 측정을 거부합니다. </li><li> 사전 및 사후 표백 이미지는 기증자 (CFP)와 수용체 (YFP) 모두에 대한 기록과 효율성이로 계산됩니다 걱정입니다 모든 D 게시&gt; D 미리 D 사전에 대한 EFF = (D 포스트 - D 미리) / D 게시물 걱정 그리고 D 게시하기 전에 각각 photobleaching 후 기증자의 강도입니다. </li><li> JMP 소프트웨어 (SAS)는 실험 다양성의 범위를 결정하는 데 사용됩니다. </li></ol> 4. 대표 결과 Transfection 효율이 30-40% 사이에 일반적으로있다. 다른 이전의 연구 결과에도 불구하고, 우리는 한 벡터의 transfection과 표현이 다른의 좋아하는 것을 보지 못했어요. 사실, 우리는 자주 키메라 또는 다른 하나 형광단의 독점적인 표현을 관찰합니다. 일반은 효율이 수용체 시스템 간의 차이가 무서워. 타이 수용체 시스템, 전형적인 값은 상피 세포에 대한 20-28% 및 내피 세포에 19-23% 있습니다. 부정적인 컨트롤, 전형적인 효율은 다음과 2~3% 있습니다. 다양성의 정도는 경험이 상당히 줄어 듭니다. <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="그림 1" /> 그림 1. transfected EA.hy 926 세포의 대표적인 이미지. EA.hy 926 세포 monolayer의) DIC 이미지. (A)에 표시되는 세포의 B) 형광 이미지. 의 C) 중첩 (A)와 (B) 시연 ~ 20~30%의 transfection 효율. <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="그림 2" /> 그림 2. 대표 수용체는 EA.hy926 감방에서 CFP와 YFP 사이에 발생하는 걱정의 분석을 사진 표백. CFP 방출 채널 (상단 패널)와 YFP 방출 채널 (아래 두 패널)은 이전에 별도의 모니터링 및 게시물 수용체가 photobleaching되었습니다. Photobleaching 실험로 제한하고 녹색 상자 내의 지역에서 계산된 값을 무서워했다. 안달 효율은 오리 엔테이션을 위해 이미지를 통합 CFP / YFP의 확대 오버레이 부족 (자주색 - 0.0)로 높은 곳에서 절대 범위 (적색 - 1.0)로 표시됩니다.

<ol>
	<li>Kim, M., Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+transmembrane+signaling+by+cytoplasmic+domain+separation+in+integrins.">Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.</a> Science. 301, 1720-1725 (2003).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipidmodified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Seegar, T. C. M., Eller, B., Tzvetkova-Robev, D., Kolev, M., Henderson, S. C., Nikolov, D. B., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie1-Tie2+interactions+mediate+functional+differences+between+angiopoietin+ligands.">Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.</a> Molecular Cell. 37 (5), 643-655 (2010).</li><li>Edgell, C. J., McDonald, C. C., Graham, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc Natl Acad Sci U S A. 80 (12), 3734-3737 (1983).</li></ol>

관심 없음 충돌 선언하지 않습니다.

성공에 중요한 몇 가지 단계가 있습니다. 그 중 가장 두드러진은 두 키메라 수용체 사이의 표현의 상대적인 수준입니다. 이 문제를 회피하기 위해 하나는 관심의 두 단백질을 표현하는 안정적인 세포 라인을 만들거나 이에 상응하는 표현을 허용하는 벡터 DNA의 최적 비율을 확인할 수 있습니다. 마찬가지로, 요리를 통해 비 균일한 transfection에 의한, 단백질 수준은 거의 세포 사이에 동등되지 않습니다. 따라서, 관심은 &#39;낮음&#39;에서 &#39;높음&#39;expressors를 구별할 지불하셔야합니다. 단백질의 표시 &#39;평균&#39;수준은 일반적으로 안정적이고 재현성 수확량 이들은 효율성을 무서워. 저희 연구소가이 문제를 해소하기 위해 추구가 하나의 방법은 &#39;도&#39;유전자 발현에 adeno 및 lenti - 바이러스를 사용하는 것입니다. 또한, 수용체 사진 표백 결정하는 다른 방법은 효율이 sensitized 방출됩니다 무서워. 실시간으로 단일 세포를 모니터링하는 sensitized 방출의 가능성에도 불구하고, 우리는 수용체 사진 표백 더 민감하고 더 신뢰할 수 발견,하지만. 마지막으로, 단백질 상호 작용을 모니터링하기 위해 안달 사용하여 도전해야하고, 멤브레인 바운드 수용체에 대한 링커 길이에주의 선택이 필요합니다. 또한, C / YFP의 추가는 종종 크게 endoplasmic reticulum과 잠돌군 Zz 장치의 집합의 단백질 발현 수준과 결과에 영향을 미칩니다. 그러나, 과도, 또는 불안 정한 상호 작용에 대한, 초조가 활용할 수있는 이상적인 방법입니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11960-069</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin- Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15070-063</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>N/A</td>
<td>&nbsp;</td>
<tr>
<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11058-021</td>
<td>&nbsp;</td>
<tr>
<td>FUGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip Dishes</td>
<td>&nbsp;</td>
<td>MatTek Corp.</td>
<td>P35G014C</td>
<td>&nbsp;</td>
<tr>
<td>pcDNA3.1(+)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>V790&#x2010;20</td>
<td>&nbsp;</td>
<tr>
<td>Mach 1 Competent Cells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C862003</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

imaging protein-protein interactions in vivo

단백질 - 단백질 상호 작용은 모든 필수 세포 프로세스의 특징입니다. 그러나 이러한 상호 작용의 대부분은 이러한 공동 immunoprecipitation과 같은 전통적인 생화 학적 방법을 통해 자신의 식별 및 분석을 방지, 과도, 또는 정력적 약한 있습니다. 이와 관련, 유전자 encodable 형광 단백질 (GFP, RFP 등) 및 형광 스펙트럼이 중복 관련은 포스터 공명 에너지 전달 (무서워) 1-3을 사용하여 생체내의 약한 상호 작용을 모니터링하는 능력을 혁신합니다. 내피 세포 표면에 수용체 - 수용체의 상호 작용을 모니터링 걱정 기반의 근접 분석의 여기, 우리는 세부의 사용을.

Watch this Scientific Journal Video about Imaging Protein-protein Interactions in vivo at JoVE.com

Imaging Protein-protein Interactions in vivo

이 프로토콜은 걱정 기반의 근접 분석을 사용하는 방법에 이미지 단백질 단백질 상호 작용을 설명합니다.

이미징 단백질 - 단백질 상호 작용 생체내에</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

imaging-protein-protein-interactions-in-vivo

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

21.2K 조회수.  Virginia Commonwealth University. 이 프로토콜은 걱정 기반의 근접 분석을 사용하는 방법에 이미지 단백질 단백질 상호 작용을 설명합니다.

비디오: Imaging Protein-protein Interactions in vivo

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

분할 Ubiquitin 기반 막 효모 두 하이브리드 (신화) 시스템 : 단백질 단백질 상호 작용을 확인을위한 강력한 도구

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

연구

생물학

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

마이크로 패턴의 표면에 단백질 - 단백질 상호 작용에 - 생체내 감지

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

담배 원형질체 및 잎에 이분자 형광 보완에 의해 시각 단백질 - 단백질 상호 작용

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

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) ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

고밀도 기능성 단백질 마이크로 어레이를 프로빙하는 단백질 - 단백질 상호 작용을 검출하기

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

를 통해 교체 단백질 - 단백질 상호 작용의 억제제의 개발 : 설계 및 개발 비 ATP 경쟁 CDK 억제제에 응용

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

고급 단백질 단백질 상호 작용 및 DNA 병 변에서 속도 론 연구에 Confocal 현미경 검사 법 기술

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...