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

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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 사항이 있습니다. 형광단 형성에 대한 분자 산소의 요구로 인해, BiFC은 산소의 존재에서 살아남기 수없는 의무를 anaerobes에서 사용할 수 없습니다. 이것은 에어로빅 생물 6 BiFC의 사용을 제한합니다. 형광단 단지의 형성은 되돌릴 수 있습니다. 이것은 다른 사람과 상호 작용하는 단백질을 방지하고 동적인 평형 6 단백질 단지의 협회와 disassociation를 중단 있습니다. 그러나,이 비가 역성은 약하거나 교통 상호 작용을 시각화하는 데 수 있습니다. 형광 단백질 조각들은 융합 아르에 대한 단백질의 독립적인 연결 제한 기능이 있습니다. 하나는 TRUE와 FALSE - 긍정적인 단백질 상호 작용 6 구별하는 데 필요한 수많은 컨트롤을 제공해야합니다. 또한, 형광단의 reconstitution가 7nm 이상의 거리에서 발생할 수로, 형광 complementation도 직접 또는 간접적으로 상호 작용을 나타낼 수 있습니다. 하나는 정확한 상호 관계 7 테스트 같은 Y2H 또는 Co - Immunoprecipitation 등 다른 방법을 사용해야합니다. 신뢰할 수있는 데이터를 생성하기 위해서는 적절한 컨트롤이 결과의 해석을 위해 사용해야합니다. 그들이 attR1과 attR2 카세트 사이의 전형적인 게이트웨이 조각을 포함하는 등 빈 벡터 직접 컨트롤로 사용할 수 없습니다. 따라서 이러한 빈 벡터 정말 &quot;빈&quot;하지 않습니다. 이 문제를 극복하기 위해, 우리는 일반적으로 컨트롤로 벡터에 융합 관련이없는 단백질 쌍을 사용합니다. 또 다른 잠재적인 문제, 특히 옛날이나 스트레스를 식물에서 식물 세포에서 자동 형광이다. 경험이 조사는 먼저 형광 배경에 익숙한 얻을 수있는 uninfiltrated 영역에서 예제를 보라 구. 최근 더 BiFC 기반 방법은 더욱 다양한 단백질의 측면, RNA 단백질 상호 작용 12, 및 DNA의 하이브리드화 13 BiFC 분석의 응용 프로그램을 확장하기 위해 개발되었습니다. 예를 들어, 여러 가지 빛깔의 BiFC 및 BiFC 기반은 살아있는 세포 14 3 원 단지를 식별하고 시각화하기 위해 개발되었습니다 (BiFC - 무서워) 분석을 무서워. 게이트웨이와 BiFC의 결합 손상 세포의 단백질 상호 작용을 이해하려는 연구자를위한 강력한 도구입니다. 우리 BiFC 시스템에 사용되는 구조는 서로 다른 조직에서 과도 표현 시스템에서 관찰되지 않을 수 있습니다 다양한 발달 단계에서 더 역동적인 단백질 상호 작용을 시각화하기 위해 안정적인 유전자 변형 식물을 생성하는 데 사용할 수 있습니다. 우리는 각각 우리 BiFC 벡터에서 H​​A 태그와 플래그 태그 (pEarleyGate201 - YC 및 pEarleyGate202 - YN)을 통합했습니다. 이 수정은 상호 작용의 시각화 후, 그러한 서부 blotting, 공동 Immunoprecipitation과 친화력 정화 등 다양한 다운 스트림 애플 리케이션을위한이 가능합니다.

<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

Nanyang Technological University

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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생물학

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 ...

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

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 ...