우리는 GPI - GFP 들어, hCD71 - GFP 구축, 그리고 다니엘 레글러, 콘스탄츠, 스위스의 대학에 대한 그녀의 친절한 도움 Katharina Strub, 스위스 제네바 대학에 대한 Quentina 비티, 린츠, 오스트리아의 대학, 감사의 말씀을 전합니다 - DAF는 건설. 과학 연구에 대한 오스트리아 연방 교육부의 GEN - AU 프로젝트,이 작품은 오스트리아 과학 기금 (프로젝트 Y250 - B03 FWF)에 의해 지원되었다.

Microcontact 인쇄 : <ol><li> 메스를 사용하여 PDMS 스탬프의 micropattern 분야를 잘라 </li><li> 에탄올 (100 %)와 증류수로 플러싱하여 micropattern가 들어있는 필드를 씻으십시오. </li><li> 질소 또는 아르곤 가스와 PDMS 스탬프를 건조시킵니다. </li><li> 피펫 50 μl BSA - Cy5 작업 솔루션 (0.67 MG / ML) 전체 micropattern 필드 솔루션으로 덮여있다 있도록 PDMS 스탬프로. 실온에서 30 분 알을 품다. </li><li> 인산과 플러싱하여 micropattern 필드를 세차 살린 (PBS)와 증류수를 버퍼. </li><li> 질소 또는 아르곤 가스와 PDMS를 건조시킵니다. </li><li> 한 에폭시 코팅 coverslip의 중간에 자체 무게에 따라 PDMS 스탬프를 놓고는 배양 접시에 실온에서 30 분 알을 품다. </li><li> coverslip의 뒷면에있는 PDMS 스탬프의 위치를​​ 분류하고 집게로 슬라이드에서 우표를 스트립. </li><li> coverslip에 microcontact - 인쇄 분야를 통해 안전한 인감 하이브 리다이 제이션 실 스틱. 레이블이 분야의 중심을 집중하는 데 도움이됩니다. </li><li> 피펫 60 μl streptavidin 작업 반응 챔버에 솔루션 (50 μg / ML)과 실온에서 60 분에 대한 샘플을 품어. </li><li> 챔버 중 하나 포트에 버퍼를 추가하고 펌프와 두 번째 포트에서 동시에 그것을 제거하여 한 ML PBS로 샘플을 씻으십시오. </li><li> 피펫 60 μl biotinylated 항체 작업 솔루션 (10 μg / 0.1 % 트윈 20과 보충 PBS의 ML, &quot;PBST&quot;) 실온에서 60 분 반응 챔버 및 부화에. </li><li> 챔버 중 하나 포트에 버퍼를 추가하고 펌프와 두 번째 포트에서 다시 제거하여 한 ML PBST하고 이후 1 ML PBS로 샘플을 씻으십시오. </li><li> 세포 시딩 준비가 될 때까지 상온에서 어둠의 PBS에서 micropatterned 표면을 저장합니다. </li></ol> micropatterned 표면에 세포의 인큐베이션 : <ol><li> 3cm 조직 문화 판의 50 % 합류로 자기편 세포를 성장. </li><li> Ethylenediaminetetraacetic 산성 (EDTA (에틸렌 다이아 민 테트라 초산)) 솔루션으로 관심을 유인 먹이 단백질을 표현하는 세포를 분리하고 1,000 rpm으로 5 분 원심 분리기. </li><li> 뜨는을 취소하고 따라 성장 매체에있는 세포 펠렛을 디졸브. </li><li> 1,000 rpm으로 5 분 원심 분리기. </li><li> 뜨는을 취소하고 적절한 성장 매체 세포 펠렛을 디졸브. </li><li> 교환 세포 현탁액의 60 μl로 micropatterned coverslip에 반응 챔버에서 PBS. </li><li> 증류수에 살균 패드를 몸으로 건조 실행 예제를 방지하기 위해 페트리 접시에 그것을 넣어 humidified 챔버를 만듭니다. </li><li> 37 박 이상의 세포를 품어 ° micropatterned 표면에 5 % CO 2 분위기에서 C. </li><li> 현미경에서 세포를 분석하기 전에, CA 2 포함한 행크의 버퍼 소금 솔루션으로 매체를 교환 +와 MG 2 +. </li></ol> 현미경 : <ol><li> coverslip는 적절한 마운트에 위치하고 세포 형태는 흰 빛 아래에서 확인합니다. </li><li> BSA - Cy5 패턴의 품질은 647 nm의 여기에 의해 확인됩니다. </li><li> 형광 먹이 단백질 (GFP 또는 YFP)의 Micropatterns는 총 내부 반사 (티르) 여기에 따라 488 또는 514 nm의에 기록됩니다. </li></ol> 대표 결과 : micropatterned 표면에 성장 세포에있는 대상 단백질 간의 상호 작용이있는 경우, 먹이가 미끼 재배포를 수행합니다. 결과 micropattern는 먹이 단백질의 형광 레이블로 시각 수 있습니다. 중요한 얻은 대비 상호 작용의 강도를 직접 측정을 (그림 1 참조)을 제공합니다. 두 단백질의 상호 작용에 따라서 간단한 평가는 가능한 것입니다 - 자세한 측정 기본 데이터를 처리하는 데 필요하지 않고. <img src="/files/ftp_upload/1969/1969fig1.jpg" alt="그림 1" /> 그림 1. 다른 상호 작용의 강점에서 라이브 셀 플라즈마 막의 미끼에 다시 정리하기. CD59 - 항체 microbiochips에 티르의 CD71 - 항체에 (A) CD71 - GFP와 transfected T24 세포의 이미지, (B) CD4와 CD4 - 항체에 cytosolic YFP 그리고 (c) GPI - GFP - DAF가 표시됩니다. 데이터에 대한 특성없는 강한 (A),도 (B) 또는 약한 상호 작용 (C). (A) (Weghuber 외., 언론), (B) (Schwarzenbacher 외., 2008)로부터 재현됩니다.

<ol>
	<li>Brameshuber, M., Schwarzenbacher, M., Kaltenbrunner, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schuetz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reply+to+"Uncoupling+diffusion+and+binding+in+FRAP+experiments.">Reply to "Uncoupling diffusion and binding in FRAP experiments.</a> Nat. Methods. 6, 183-184 (2009).</li><li>Kim, P. W., Sun, Z. J., Blacklow, S. C., Wagner, G., Eck, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Zinc+Clasp+Structure+Tethers+Lck+to+T+Cell+Coreceptors+CD4+and+CD8.">A Zinc Clasp Structure Tethers Lck to T Cell Coreceptors CD4 and CD8.</a> Science. 301, 1725-1728 (2003).</li><li>Ramm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+screening:+a+technology+in+transition.">Image-based screening: a technology in transition.</a> Curr. Opin. Biotechnol. 16, 41-48 (2005).</li><li>Schwarzenbacher, M., Kaltenbrunner, M., Brameshuber, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+for+quantitative+analysis+of+protein-protein+interactions+in+living+cells.">Micropatterning for quantitative analysis of protein-protein interactions in living cells.</a> Nat. Methods. 5, 1053-1060 (2008).</li><li>Weghuber, J., Brameshuber, M., Sunzenauer, S., Lehner, M., Paar, C., Haselbruebler, T., Schwarzenbacher, M., Kaltenbrunner, M., Paster, W., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein-protein+interactions+in+the+live+cell+plasma+membrane+by+quantifying+prey+redistribution+upon+bait+micropatterning.">Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.</a> Methods in Enzymology. , .</li></ol>

비디오 시각화 연구실의 홈페이지에서 가져온 올림푸스, 브라운 대학에 의해 제공 또는 (김 외., 2003)에서 재현되었습니다 이미지가 포함되어 있습니다.

함께 비디오 생활 세포의 플라즈마 - 멤브레인 (.; Brameshuber 외, 2009;.. Weghuber 외, 언론에서 Schwarzenbacher 외, 2008)에서 단백질 - 단백질 상호 작용을 검출하는 방법을 보여줍니다. 원칙적으로, 모든 TIRF 기반 현미경 플랫폼은 판독 시스템으로 사용할 수 있습니다. 높은 감도 (단일 분자의 검출에 대한 예) 원하는 경우에만, 고급 현미경이 필요합니다. 최상의 결과가 준비 과정에서 다음과 같은 중요한 사항을 달성하기 위해서는 특별한주의가 필요합니다 : <ol class="lroman"><li> 어두운 조건 스토어 Cy5 주식 솔루션은 형광단의 표백 방지와 microcontact 인쇄하기 전에 신선한 BSA - Cy5 작업 솔루션을 준비합니다. </li><li> 피펫 팁로 PDMS에 micropattern을 긁거나 형광단의 표백 피하기 위해 어두운 조건에서 샘플을 부화하지 않도록주의하십시오. </li><li> 페트리 접시에 유리 슬라이드의 고집 방지하기 위해 멸균 패드에 coverslip 놔. PDMS 스탬프는 유리 슬라이드에 준수되면, 그것을 이동하지 않습니다. </li><li> 반응 챔버에서 공기 방울을 피하고 형광단의 표백 피하기 위해 어두운 조건에서 샘플을 품어. </li><li> 소금 결정의 형성을 피하기 위해 2 개 이상 분 샘플 건조시키지 마십시오. </li><li> 세포의 분리에 관해서는 트립신을 사용하지 않는 그것 클리브은 지금까지 표현 막 단백질의 세포외 도메인 것입니다. 트립신을 피하기 것은 바로 표면에 세포 부착 후 micropatterns을 형성 낮은 회전율 속도로 단백질을 위해 도움이 될 것입니다. </li><li> 현미경에 incubated 세포의 수를 제어하고 세포가 이상 30-50%의 합류에 도달하지 않은지 확인하십시오. 과도한 세포 인해 항체 - 코팅 표면에 세포의 빠른 첨부 파일에 즉시 제거되었는지 확인합니다. </li><li> 고품질 BSA - Cy5 패턴만을 슬라이드가 추가 분석을 위해 사용될 수 있습니다. </li><li> 형광 먹이 단백질의 안정적인 표현으로 인해 스캔마다 분석할 수 세포의 더 많은 것이 바람직합니다. </li><li> 적절한 TIRF 조정은 cytosolic 배경으로 인해 패턴을 시각화하기 위해 필수입니다. </li></ol> 마이크로 patterning 기술은 단백질 - 단백질 상호 작용의 분석을위한 몇 가지 가능성을 제공합니다. 첫째, 지역, 공간 해결 단백질 단백질 상호 작용의 부량와 함께 또한 약한 또는 간접적으로 상호 작용의 검출은 잘못된 반응 또는 제외 높은 번호를 부여의 단점없이 가능합니다. 둘째 그것은 2 하이브리드 화면과 같은 생화 학적 방법에 의해 달성하기 어렵습니다 라이브 세포의 플라즈마 막에 단백질 단백질 상호 작용을 분석하기 위해 연구자 수 있습니다. 셋째 방법은 온도, 다른 단백질이나 다른 분자 또는 사후 translational 수정의 존재와 같은 환경 변화에 의해 변조된 아르 미끼 - 먹이 상호 작용의 검출을 허용합니다. 따라서 분석 살 세포의 맥락에서 주어진 상호 작용 쌍 심사 변조기를 허용합니다. 또한 캡처 리간드 또는 monovalent 리간드의 사용의 표면 밀도를 줄임으로써, 휴식 상태의 분석이 가능하게됩니다. 적절한 검색 플랫폼을 사용하는 경우 마지막으로, 분석 세포의 숫자는 약물 검사를위한 제약 회사의 높은 처리량 요구 (램, 2005)와 일치 정도로 높습니다.

in-vivo detection of protein-protein interactions on micro-patterned surfaces

분자의 상호 작용 네트워크를 무너지고있다<em&gt;에 - 생체내</em&gt; 세포 기능과 신진 대사를 조절하는 메커니즘을 이해하는 열쇠입니다. 세포의 분자 상호 작용을 해결하기위한 방법 론적 선택의 무리가 개발하지만, 이러한 방법의 대부분은 거의 양적 오히려 간접 따라서되는 고통되었습니다. 반대로, 몇 가지 고급 양적 접근 방법이 도입되었다, 그러나 높은 처리량을 확장하기가 어렵습있는. 양적 정보를 추출 가능성이 높은 처리 기능을 결합하기 위해, 우리는 최근에 단백질 단백질 상호 작용을 파악하기위한 새로운 개념을 (Schwarzenbacher 개발<em&gt; 외</em&gt;., 2008). 여기, 우리는 ( &quot;미끼&quot;) 형광단 - 라벨 단백질 ( &quot;먹이&quot;)와 막 단백질 사이의 상호 작용을 분석하는 허용이 시스템의 설계 및 건설에 대한 자세한 프로토콜을 설명<em&gt;에 - 생체내</em&gt;. 세포는 미끼 exoplasmic 도메인에 대한 항체로 작용 micropatterned 표면에 도금입니다. 미끼 - 먹이 상호 작용은 형광등 먹이의 재배포를 통해 assayed 있습니다. 방법은 선별 도구로 그것이 적용하고, 아래로 단일 분자 수준 높은 감도, 약한 상호 작용을 감지하는 능력, 높은 처리 능력에 의해 특징입니다.

Watch this Scientific Journal Video about In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces at JoVE.com

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

이 비디오는 마이크로 패턴 표면의 사용에 의해 단백질 단백질 상호 작용의 후속 분석 실험을 보여줍니다. 접근 방식은 살아있는 세포의 단백질 상호 작용을 감지하는 가능성을 제공하며 양적 정보를 추출 가능성이 높은 처리 기능을 결합한 제품입니다.

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 ...

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

10.7K 조회수.  Johannes Kepler Universitat Linz. 이 비디오는 마이크로 패턴 표면의 사용에 의해 단백질 단백질 상호 작용의 후속 분석 실험을 보여줍니다. 접근 방식은 살아있는 세포의 단백질 상호 작용을 감지하는 가능성을 제공하며 양적 정보를 추출 가능성이 높은 처리 기능을 결합한 제품입니다.

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

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 ...

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Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

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, ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Spectrally 해결된 두 광자 현미경을 사용하여 G 단백질 결합 수용체 상호 작용의 생체내 부량에서

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

을 바탕으로 단백질 - 단백질 상호 작용의 확인에 프로토콜<sup&gt; 15</sup&gt; N 신진 대사 라벨링, Immunoprecipitation, 수량 질량 분석법 및 선호도 변조

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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 ...

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. ...