저자는이 장치를 설계하는 과정 동안 도움이 토론 살만 Khetani, 제라드 앨런, 크리스 플레임, 오스틴 Derfus 감사드립니다. 이 작품은 국립 과학 재단 (National Science Foundation) 학부 조기 경력 개발 프로그램, 당뇨병과 소화기 및 신장 질환 보건 / 국립 연구소 국립 연구소, 그리고 데이비드와 루실 패커드 재단에 의해 지원되었다. 에이는 루스 L. Kirschstein 내셔널 리서치 서비스 상에 의해 지원되었다.

세포 배양의 준비 : <ol><li> 플라즈마 처리 폴리스티렌과 코팅 실리콘 부품과 함께 시작합니다. </li><li> 적절한 세포외 기질 단백질로 코트 부품 원하는 세포 유형의 첨부 파일을 지원합니다. hepatocytes 들어, 37 50 G / ML 콜라겐 - 1 솔루션 ° 45 분 들면 C에서 알을 품다. 3T3 섬유아 세포 들어, 매트릭스가 필요하지 않습니다. </li><li> 연락처 구성에 보완 부분 부분을 잠금. </li><li> 소독 10 분 최소 70 %의 에탄올에 만끽해보세요. 세포 배양 매체 ddH 2 O 및 1X의 2 배 린스. </li><li> 적절한 문화 매체 500,000 셀 / ML의 농도에 씨앗 세포. 12 잘 문화 판의 각 잘 1 ML을 사용합니다. </li><li> 균등하게 다시 정지 세포마다 15 분 흔들어, 1 시간을위한 부화. </li><li> 합류 monolayer 1 시간 이후에 달성되지 않은 경우, 새로운 서스펜션으로 세포 현탁액 및 반복 시딩을 대기음. 원하는 세포 밀도를 얻을 때까지 반복합니다. </li><li> 보완 부분을 제거합니다. 신선한 우물에 부품을 전송하고 세포가 완전히을 준수하고 확산 수 있도록 밤새 품어. </li><li> 간격이나 연락처 구성 중 하나에 해당하는 부분을 잠금으로 양식 공동 문화. 구성은 문화, 동안 원하는 시점에서 변경될 수 있습니다. </li><li> 미디어를 변경하는 경우, 가능 한한 짧은 시간에 대한 세포 건조 떠나 선호 잠시 한두 돌봐. 한 손으로 액체를 밖으로 꺼내는 즉시 한편을 사용하여 미디어를 대체합니다. </li></ol> 재사용을위한 처리 실리콘 부품 : <ol><li> 표백에 몸을 담글 수와 물로 rinsing하여 세포를 스트립. </li><li> 부품이 완전히 건조하도록 허용합니다. 폴리스티렌을 벗겨 2 H에 대한 톨루엔의 만끽해보세요. </li><li> 120 ° C.에 가열 : 피라 냐는 물고 기지 너한테 솔루션 (H 2 SO 4 1시 2분 H 2 O 2)에서 클린 </li><li> ddH 2 O.의 지속적인 흐름에 따라 15 분 린스 즉시 폴리스티렌을 입금하지 않을 경우, 물에 부분을 저장합니다. </li><li> 100 MG / ML에서 톨루엔의 폴리스티렌을 디졸브. 1 시간에 대한, 또는 완전히 해산까지를위한 폴리 프로필렌 원뿔의 소용돌이. 솔루션의 조금 더보다 1 ML은 매 10 부품이 필요합니다. </li><li> 30 초에 대한 2,​​400 rpm으로 각각의 실리콘 부품에 스핀 코트의 폴리스티렌 솔루션입니다. </li><li> 120 ° C. 적어도 5 H에 대한 베이크 </li><li> 1 분에 대한 산소 플라즈마 (200 mTorr, 200 W)로 처리합니다. </li></ol>

<ol>
	<li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proceedings of the National Academy of Sciences. 104, 5722-5726 (2007).</li><li>Bhatia, S. N., Balis, U. J., Yarmush, M. L., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cell-cell+interactions+in+preservation+of+cellular+phenotype:+cocultivation+of+hepatocytes+and+nonparenchymal+cells.">Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells.</a> The FASEB Journal. 13, 1883-1900 (1999).</li><li>El-Ali, J., Sorger, P. K., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+on+Chips.">Cells on Chips.</a> Nature. 442, 403-411 (2006).</li></ol>

The authors have nothing to disclose.

이 시스템은 동적으로 세포 수준에서 조작하는 조직의 공간적 조직을 가능하게한다는 점에서 고유합니다. 따라서,이 장치는 세포 신호 역학, 연락처 - 중재 비교 용해 신호, 세포의 운명 결정, 독성학, 그리고 세포 누화와 같은 주제를 걸​​쳐, 새로운 생물 학적 실험의 숫자를 사용할 수 있습니다. 문화 기판 표준 조직 문화 플라스틱이기 때문에이 장치가 널리 generalizable되어야하고, 시스템은 표준 방식과 문화 ...

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<td>Silicon Comb Substrates</td>
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<td>Parts are available for collaborative research projects. Please contact Elliot Hui (eehui @ alum . mit . edu) or Sangeeta Bhatia (sbhatia @ mit . edu).</td>
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silicon microchips for manipulating cell-cell interaction

세포 microenvironment의 역할은 개발에서 악성 변화로 거의 모든 포유 동물 조직의 세포 운명과 기능을 결정하는 중요한로 인식되고 있습니다. 특히, 이웃 기질과 상호 작용은 생물 학적 현상의 과다에 연루되어 있지만, 종래의 기술은 이러한 상호 작용의 공간과 동적 요소를 심문할 수있는 능력을 제한하고 있습니다. Micromechanical 재구성 문화 (RC), 우리는 동적 기계 위치 조정을 통해 셀 - 세포 상호 작용을 제어하는​​ 부품을 움직이는 micromachined 실리콘 기판을 사용합니다. 이전,이 방법은 시간 의존 상호 작용과 용해 신호 1 제한된 범위를 보여주는, hepatocytes 및 비 - parenchymal 세포의 공동 문화에 세포 통신을 조사하기 위해 적용되었습니다. 여기서는 자세히 RC 시스템의 준비와 사용에 대해 설명합니다. 우리는 간격 및 연락처 구성 사이의 작동을 포함하여 핀셋을 사용하여 장치 부품의 처리를 시연하여 시작 (세포 인구가 좁은 80 μm의 간격으로 구분하여, 또는 직접 친밀한 접촉). 다음, 우리는 자세히 문화 기판을 준비하는 과정, 그리고 다단계 셀 시딩 프로세스가 합류 세포 monolayers를 얻기 위해 필요합니다. 라이브 현미경을 사용하여, 우리는 다음 다른 가능한 실험 구성 사이 세포의 실시간 조작을 설명합니다. 톨루엔과 피라 냐는 물고 기지 너한테 청소, 폴리스티렌 코팅 및 산소 플라즈마 처리 : 마지막으로, 우리는 재사용을위한 장치 표면을 생성하기 위해 필요한 단계를 보여줍니다.

Watch this Scientific Journal Video about Silicon Microchips for Manipulating Cell-cell Interaction at JoVE.com

Silicon Microchips for Manipulating Cell-cell Interaction

이 문서는 마이크로 미터 규모의 자기편 세포 사이의 세포 - 세포 상호 작용의 동적 규제를위한 실험적인 접근 방법을 설명합니다. hepatocytes와 stromal 세포 사이의 세포 의사 소통의 조작이 증명됩니다. 개발 플랫폼 개발 및 pathogenesis을 포함한 생물 학적 과정의 다양한 세포 - 세포 상호 작용의 조사를 수 있습니다.

세포 세포 상호 작용을 조작에 대한 실리콘 마이크로 칩

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

silicon-microchips-for-manipulating-cell-cell-interaction

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10.7K 조회수.  MIT - Massachusetts Institute of Technology. 이 문서는 마이크로 미터 규모의 자기편 세포 사이의 세포 - 세포 상호 작용의 동적 규제를위한 실험적인 접근 방법을 설명합니다. hepatocytes와 stromal 세포 사이의 세포 의사 소통의 조작이 증명됩니다. 개발 플랫폼 개발 및 pathogenesis을 포함한 생물 학적 과정의 다양한 세포 - 세포 상호 작용의 조사를 수 있습니다.

비디오: Silicon Microchips for Manipulating Cell-cell Interaction

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

체외에서 분리된 뉴런을 조작에 대한 레이저 족집게를 사용하여

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

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 ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

단백질 정제에서 사용하기 위해 자동화된 소수성 상호 작용 크로마 토그래피 칼럼 선정

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

염색질 상호 작용을 매핑 및 전사 규정을 이해하는 데 이점 엔드 태그 장면 (ChIA-PET)과 염색질의 상호 작용 분석

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

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

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

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

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

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

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

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

쥐의 눈물샘을 조작

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

조사 단백질 기능, 시공간 현지화 및 단백질 상호 작용 네트워크를위한 유도 LAP-태그 안정 세포주

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase 보완성 이미징 분석 결과 Nicotiana benthamiana 잎 뚜렷이 결정 단백질 단백질 상호 작용 역동성에 대 한

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...