Yazarlar Salman Khetani Jared Allen, Chris Flaim ve Austin Derfus bu cihaz tasarımı sürecinde yararlı tartışmalar için teşekkür ederiz. Bu çalışma, Ulusal Bilim Vakfı Fakültesi Erken Kariyer Geliştirme Programı Sağlık / Ulusal Diyabet ve Sindirim ve Böbrek Hastalıkları Enstitüsü, Ulusal Sağlık Enstitüleri ve David ve Lucile Packard Vakfı tarafından desteklenmiştir. Eeh bir Ruth L. Kirschstein Ulusal Araştırma Hizmet Ödülü tarafından desteklenmiştir.

Hücre kültürlerinin hazırlanması: <ol><li> Plazma ile tedavi edilen polistiren kaplamalı silikon parçaları ile başlayın. </li><li> Uygun ekstraselüler matriks proteinleri ile Coat parçaları istenilen hücre tipi eklenti desteği. Hepatositler için, 37 ° 50 g / ml Kollajen-1 çözümü ° C de 45 dakika inkübe edin. 3T3 fibroblastlar için, herhangi bir matris ihtiyaç vardır. </li><li> Tamamlayıcı parçaları ile temas yapılandırma parça kilitleyin. </li><li> Sterilize etmek için 10 dakika en az% 70 etanol içinde bekletin. Hücre kültür ortamı GKD 2 O, 1x 2x durulayın. </li><li> Tohum hücrelerinin uygun kültür ortamı 500.000 hücre / ml &#39;lik bir konsantrasyon. 12-iyi bir kültür plaka her kuyuda 1 ml kullanın. </li><li> Eşit olarak yeniden askıya hücreler her 15 dakikada bir sallayarak, 1 saat süreyle inkübe edin. </li><li> Konfluent tek tabaka 1 saat sonra elde edilmemiş ise, hücre süspansiyonu ve taze bir süspansiyon ile tekrar tohumlama aspirat. Istenilen hücre yoğunluğu elde edene kadar tekrarlayın. </li><li> Tamamlayıcı parçaları sökün. Taze kuyu parça transfer ve hücrelerin tam olarak uymak ve yaymak için izin gece boyunca inkübe edin. </li><li> Boşluk veya kontakt konfigürasyonu birine uygun parçaları kilitleme Formu ortak kültürler. Yapılandırma sırasında herhangi bir kültür istenilen noktada değiştirilebilir. </li><li> Medya değiştirirken, mümkün olan en kısa sürede hücreleri kuru bırakmak tercihen sadece bir ya da iki saniye ilgilenir. Tek elle sıvı çizin ve diğer yandan kullanarak medya hemen değiştirin. </li></ol> İşleme yeniden kullanım için silikon parçaları: <ol><li> , Çamaşır suyu iliklerine ve su ile durulama hücreleri soyun. </li><li> Parçalar tamamen kurumasını bekleyin. Toluende polistiren şerit 2 saat bekletin. </li><li> 120 ° C ısıtılmış: piranha çözümü (H 2 SO 4 01:02 H 2 O 2) Temiz </li><li> GKD 2 O sürekli bir akış altında 15 dakika süreyle yıkayın Polistiren hemen yatırmak değilseniz parçaları, su saklayın. </li><li> 100 mg / ml toluen polistiren çözülür. Polipropilen konik, yaklaşık 1 saat ya da tamamen eriyene kadar karıştırın. Çözüm biraz fazla 1 ml, her 10 parça için gereklidir. </li><li> 30 sn için 2400 rpm&#39;de bireysel silikon parçaları Spin kat polistiren çözüm. </li><li> 120 ° C&#39;de en az 5 saat süreyle pişirin </li><li> 1 dakika boyunca oksijen plazma (200 mTorr, 200 W) ile tedavi. </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.

Bu sistem, dinamik hücresel seviyede manipüle doku mekansal organizasyon sağlayan benzersiz. Sonuç olarak, bu cihaz, hücrelerarası sinyalizasyon dinamikleri, iletişim aracılı karşı çözünür sinyal, hücre kaderinin kararları, toksikoloji ve hücresel crosstalk gibi konuları kapsayan bir dizi yeni biyolojik deneyler sağladı. Bu cihaz yaygın kültür substratı standart doku kültürü plastik olduğundan genellenememektedir olmalıdır ve sistemi standart kültür yöntemleri ve testleri ile uyumlu. Bu nedenle, biz bu plat...

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		<th>Material Name</th>
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<td>Silicon Comb Substrates</td>
<td>Tool</td>
<td>N/A</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

Hücresel mikroçevresinin geliştirme malign transformasyon hemen hemen tüm memeli dokularında hücre kaderi ve fonksiyon belirlenmesinde önemli bir rol olarak kabul edilmektedir. Özellikle, çevre stroma ile etkileşim biyolojik olayların bir bolluk suçlanmıştır, ancak geleneksel teknikler bu etkileşimlerin mekansal ve dinamik elemanlar sorgulamak için sınırlayabilir. Mikromekanik Reconfigurable Kültür (RC), hareketli parçaları ile mekanik yeniden konumlandırma sayesinde hücre-hücre etkileşimleri kontrol etmek için dinamik bir Mikroişlenmiş silikon taban istihdam. Daha önce, bu yöntem, zamana bağımlı olarak çözünür sinyalizasyon 1 etkileşimleri ve sınırlı gösteren, hepatosit ve olmayan parankimal hücrelerin ortak kültürler hücrelerarası iletişimi araştırmak için uygulanmıştır. Burada, RC sistemi ayrıntılı olarak hazırlanması ve kullanımı açıklanır. Biz boşluğu ve kontakt yapılandırmaları arasında tahrik de dahil olmak üzere, cımbız kullanarak aygıt parça işleme göstererek başlar (hücre popülasyonlarının 80 mikron dar bir boşlukla ayrılmış, ya da doğrudan yakın temas halinde). Sonra, kültür substratları hazırlama sürecinin ayrıntılı ve çok adım hücre Tohumlama işlemi konfluent hücre mono tabakaları elde etmek için gerekli. Canlı mikroskobu kullanarak, biz o zaman farklı olası deneysel yapılandırmaları arasında hücreler gerçek zamanlı manipülasyon göstermektedir. Toluen ve piranha temizlik, polistiren kaplama ve oksijen plazma tedavisi: Son olarak, biz, yeniden kullanım için cihaz yüzeyi yeniden için gerekli adımları göstermektedir.

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

Silicon Microchips for Manipulating Cell-cell Interaction

Bu makale, mikrometre ölçeğinde yapışık hücreleri arasındaki hücre-hücre etkileşimleri dinamik düzenlenmesi için deneysel bir yaklaşımı açıklar. Manipülasyon, hepatosit ve stromal hücre arasındaki hücrelerarası iletişimi gösterilmiştir. Geliştirilen platform geliştirme ve patogenezinde de dahil olmak üzere çeşitli biyolojik süreçlerin, hücre-hücre etkileşimleri soruşturma sağlar.

Hücre hücre Etkileşim kurgulama için Silikon mikroçipler

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

Research

JoVE Journal

Biology

10.7K Görüntüleme.  MIT - Massachusetts Institute of Technology. Bu makale, mikrometre ölçeğinde yapışık hücreleri arasındaki hücre-hücre etkileşimleri dinamik düzenlenmesi için deneysel bir yaklaşımı açıklar. Manipülasyon, hepatosit ve stromal hücre arasındaki hücrelerarası iletişimi gösterilmiştir. Geliştirilen platform geliştirme ve patogenezinde de dahil olmak üzere çeşitli biyolojik süreçlerin, hücre-hücre etkileşimleri soruşturma sağlar.

Video: 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.

In Vitro İzole Nöronlar kurgulama için Lazer Cımbız

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

Biyoloji

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)

Helios Gen Tabancası kullanarak Soğan Hücrelerde Protein-Protein Etkileşimi Bimoleküler Floresan tamamlama (BiFC) Testi

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 and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

In vivo ve in vitro Adaptör-clathrin Etkileşim Çalışmaları

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

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.

Protein Saflaştırma Kullanım için Otomatik Hidrofobik Etkileşim Kromatografisi Sütun Seçimi

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.

Kromatin Etkileşimler haritalama ve Transkripsiyon Yönetmelik anlamak için paired-end Tag Dizileme (Chia-PET) ile Kromatin Etkileşim Analizi

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.

Düşük afiniteli Ekstrasellüler Reseptör-ligand Etkileşimlerinin Ölçeklenebilir Algılama için Avidite tabanlı Ekstrasellüler Etkileşim Gösterimi (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.

Protein-Protein Etkileşimi Okumak Manyetik Boncuk Kullanarak Bir Sıvı Faz Affinity Yakalama Testi: Poliovirüs-Nanobody Örnek

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.

Kemirgen Gözyaşı bezi kurgulama

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.

Peptit Diziler protein-protein etkileşim Siteleri belirlenmesi

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.

Incelenmesi Protein Fonksiyonu, Spatiotemporal Yerelleştirme ve Protein Etkileşim Ağları için İndüklenebilir LAP-etiketli Kararlı Hücre Hatları

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