우리는 원고와 일반적인 실험실 지원을 읽을 바이올렛 Nguyen 감사합니다.

1. Agrobacterium 문화의 준비 <ol><li> Agrobacterium 긴장 GV3101은 이전에 게이트웨이와 호환 BiFC 벡터 pEarleyGate201 - YC 및 pEarleyGate202 - YN, 융합 GOI의 구조가 포함된 각 변형. </li><li> BiFC 융합 단백질의 5 ML YEB (5g L -1 쇠고기 추출물, 1g L -1 효모 추출물, 5g L -1 펩톤, 5g L -1 자당, 2mM MgSO 4, 산도 7.2) 문화를 예방하기와 agrobacterium 변형 구성 적절한 항생제 선택합니다. 28 야간 문화를 성장 ° C 200 rpm으로 흔들어. </li><li> 멸균 1.5 ML의 원심 관에 문화 1 ML를 제거합니다. </li><li> 상온에서 10 분 1,000g에서 펠릿 세포가 뜨는 폐기. </li><li> 침투 미디어 1 ML (5g L -1 D - 글루코오스, 50 MM MES (시그마), 2 MM 나 3 PO 4, 0.1 MM acetosyringone) 11을 추가하여 펠렛을 씻으십시오. pipetting하여 Resuspend 펠렛, 10 분 1,000g에서 다시 펠릿 세포. </li><li> 세척 단계 두 번 더 반복합니다. </li><li> 0.5 ML의 침투 미디어의 펠렛을 Resuspend. </li><li> 600 nm의 흡광도에서를 측정하고 1.2-1.0로 OD 600 최종 조정합니다. </li><li> 새로운 1.5 ML 튜브에 각 구조의 agrobacterium 정지의 동일한 금액을, 10 초위한 와동 나누어지는. 하나의 침투에 대한 각 구조 100 μL 정도 이상입니다. </li><li> agrobacterium 혼합물은 이제 침투 준비가되었습니다. </li></ol> 2. 침투 <ol start="11"><li> N. benthamiana 공장 16 - H 조명 및 8 - H 어두운 사이클로, 21 ° C에서 성장하고 있습니다. 6 주 된 식물 다섯은 침투에 대한 사용해야합니다. </li><li> stomata 완전히 열 수 있도록 infiltratring 전에 1 H에 대한 하얀 빛 아래 성장 공간과 장소에서 식물을 제거합니다. </li><li> 1 - ML 슬립 팁 투베르쿨린 주사기 바늘없이 resuspended agrobacterium 혼합을 그립니다. </li><li> 잎의 아래쪽에 대해 주사기의 팁을 놓고 직접 손가락으로 잎의 상단을 지원하면서 부드럽게 플런저를 우울. 그것이 mesophyllar 공기 공간을 가득 채우고 같은 액체는 나뭇잎을 통해 확산됩니다. </li><li> 미래를 식별 마커 펜으로 침투 지역을 라벨. </li><li> 다른 구조로 침투하면 장갑을 변경하거나 infiltrations 사이에 70 % 에탄올과 그들을 청소하거나 있는지 확인하십시오. 가능하면, 교차 오염을 방지하기 위해 다른 샘플 간의 잎의 중륵맥 공간을 둡니다. </li><li> 정상적인 성장 조건에서 성장 캐비닛에 식물을 놓습니다. </li></ol> 3. 재구성 YFP 신호의 관측 <ol start="18"><li> 침투 영역 내에서 잎 조직의 소비세 5mm X 5mm 세그먼트 </li><li> 물 속에 들어가면, 유리 현미경 슬라이드에 coverslip으로 샘플을 커버하고 공촛점 또는 형광 현미경을 사용하여 상호 작용을 조사, 샘플을 탑재합니다. </li><li> 시간 코스 이상의 여러 다른 위치를 표시하는 형광 융합 단백질을 높일 수 표현 / 매매 이상과 같은 표현식은 최대 5 일 후로 침투의 시간에서 24 시간마다 모니터링해야합니다. </li></ol> 4. 대표 결과 : BiFC 분석하여 테스트 단백질 단백질 상호 작용의 예제는 그림 1에 표시됩니다. AtTHP1과 AtSAC3A는 효모 TREX - 2 복합 Arabidopsis 상동체의 구성 요소로 여겨지고 있습니다. 그들은 nuclearporin AtNUP1과 상호 작 용할 수 있으며 mRNA 수출 10 용이하게하실 수 있습니다. AtNUP1가 pEarlyGate202 - YN에 복제하는 동안 AtTHP1와 AtSAC3A는 pEarlyGate201 - YC으로 복제했다. 구조는 이후 GV3101으로 변형되었다. 침투에 대한 (AtNUP1 + AtSAC3A,, AtTHP1 + AtSAC3A AtTHP1 + AtNUP1) 세 개의 구성 가능한 조합은 3와 쌍을했다. 재구성 YFP 신호 침투 후 LCSM 48시간 아래에 관찰되었다. 신호는 표피 세포에서 관찰하고 이러한 단백질의 서브 셀룰러 로컬 리 제이션과 일치 핵 주변이나 핵 플라즈마에 현지되었습니다. 부정적인 통제는 YFP 신호를 보여주지 않았다. <img src="/files/ftp_upload/3473/3473fig1.jpg" alt="그림 1" /> 그림 1. Arabidopsis TREX - 2 mRNA 수출 복잡한 구성 요소가 서로 상호 작용할 수 있습니다. N. benthamiana 단풍, GOI의 구조와 공동 변화는 Leica TCS SP2 confocol 현미경을 사용하여 침투 후 48-72 H 몇 군데 있었다 pEarleyGate201 - YC 및 pEarleyGate202 - YN (로 표시)로 융합된. 이미지 통합 공촛점 YFP 및 표피 N.의 밝은 필드 이미지로 표시됩니다 benthamiana 잎 세포

<ol>
	<li>Ohad, N., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+bimolecular+fluorescence+complementation+(BiFC)+to+assay+protein-protein+interaction+in+plants.">Utilizing bimolecular fluorescence complementation (BiFC) to assay protein-protein interaction in plants.</a> Methods in molecular biology. 655, 347-358 (2010).</li><li>Fang, Y., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiFC+imaging+assay+for+plant+protein-protein+interactions.">BiFC imaging assay for plant protein-protein interactions.</a> Cold Spring Harbor protocols. 2, (2010).</li><li>Schutze, K., Harter, K., Chaban, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+to+study+protein-protein+interactions+in+living+plant+cells.">Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells.</a> Methods in molecular biology. 479, 189-202 (2009).</li><li>Hiatt, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+analysis+of+protein+interactions+in+Caenorhabditis+elegans.">Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans.</a> Methods. 45, 185-191 (2008).</li><li>Barnard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+implementation+of+split-GFP-based+bimolecular+fluorescence+complementation+(BiFC)+assays+in+yeast.">Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast.</a> Biochemical Society Transactions. 36, 479-482 (2008).</li><li>Kerppola, T. 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관심 없음 충돌 선언하지 않습니다.

BiFC 분석은 단백질 상호 작용을 공부를위한 강력한 도구입니다. 전통 Y2H 분석과는 달리, BiFC뿐만 아니라 단백질 단백질 상호 작용의 시각화을 허용뿐만 아니라 하위 세포 지방화 같은 단백질 복잡한 더 많은 정보를 제공합니다. 또한, 그것은 두 후보 단백질이 세번째 파트너 가까이에 충분히 가져 수있는만큼 간접적인 상호 작용을 검출하는 것이 가능합니다. 모든 기술과 마찬가지로, BiFC는 제한 ?...

<table border='1'>
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma</td>
			<td>M2933</td>
		</tr>
		<tr>
			<td>1 mL slip-tip tuberculin syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
	</tbody>
</table>

detection of protein interactions in plant using a gateway compatible bimolecular fluorescence complementation (bifc) system

우리는 생체내 두 단백질 사이의 상호 작용을 테스트하는 BiFC 기술을 개발했습니다. 이 두 아닌 중복 조각으로 분할 노란색 형광 단백질 (YFP를)에 의해 수행됩니다. 각 조각은 관심의 유전자에 프레임에 복제됩니다. 이러한 구성은 다음 융합 단백질의 통과 식을 수 있도록 Agrobacterium의 중재 ​​변환을 통해 Nicotiana benthamiana에 공동 변형하실 수 있습니다. 심리 단백질은 1-7을 상호 작용을 때 YFP 신호의 reconstitution에만 발생합니다. 단백질 단백질 상호 작용을 테스트하고 유효성을 검사하려면 BiFC는 효모 두 하이브리드 (Y2H) 분석과 함께 사용할 수 있습니다. 이것은 Y2H에서 간과된 수있는 간접적인 상호 작용을 검색할 수 있습니다. 게이트웨이 기술에 관심 셔틀의 유전자를 (GOI)에 연구자 수있는 범용 플랫폼입니다 많은 표현 가능한 8,9와 같은 기능 분석 시스템과 같은. 오리 엔테이션 및 읽기 프레임 모두 표현 가능한 클론을 만들기 위해 제한 효소 또는 결합을 사용하지 않고 유지하실 수 있습니다. 그 결과, 하나는 실험을 통해 일관성있는 결과를 보장하기 위해 모든 다시 시퀀싱 단계를 제거할 수 있습니다. 우리는 BiFC와 Y2H 모두 assays 10을 수행하기 위해 사용하기 쉬운 도구를 통해 연구자를 제공 게이트웨이 호환 BiFC 및 Y2H 벡터 일련의를 만들었습니다. 여기, 우리는 N.에 단백질 단백질 상호 작용을 테스트하기 위해 BiFC 시스템을 사용의 용이성을 보여주 benthamiana 식물.

Watch this Scientific Journal Video about Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System at JoVE.com

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

우리는 공장에서 단백질 - 단백질 상호 작용을 테스트하는 기술을 개발했습니다. 노란색 형광 단백질 (YFP은) 2 비 중복 조각으로 분할합니다. 각 조각은 융합 단백질의 표현을 활성화, 게이트웨이 시스템을 통해 관심의 유전자에 프레임에 복제됩니다. 심리 단백질이 상호 작용을 때 YFP 신호의 Reconstitution에만 발생합니다.

게이트웨이 가능 Bimolecular의 형광 Complementation (BiFC) 시스템을 사용하여 공장에서 단백질 상호 작용의 검출

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

detection-protein-interactions-plant-using-gateway-compatible

Research

JoVE Journal

Biology

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

25.2K 조회수.  University of Western Ontario. 우리는 공장에서 단백질 - 단백질 상호 작용을 테스트하는 기술을 개발했습니다. 노란색 형광 단백질 (YFP은) 2 비 중복 조각으로 분할합니다. 각 조각은 융합 단백질의 표현을 활성화, 게이트웨이 시스템을 통해 관심의 유전자에 프레임에 복제됩니다. 심리 단백질이 상호 작용을 때 YFP 신호의 Reconstitution에만 발생합니다.

비디오: Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

자기 변조 Biosensing 시스템을 사용하여 생물 학적 Assays의 급속한 동차 검색

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

연구

생물학

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 ...

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 ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Bimolecular 형광 Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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 ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

이분자 형광 보완의 유세포 분석 : 단백질 - 단백질 상호 작용을 연구하는 높은 처리량 정량 방법

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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 ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

시차 주사 Fluorimetry를 사용하여 단백질 - 리간드 상호 작용의 결정

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

형광 단백질 분해 효소 보호를 사용하여 막 단백질 토폴로지를 결정 (FP​​P)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...