Bu çalışma NIH hibe programı R01NS046835 ve R01AG034389 ve Dimes hibe 6FY08-322 Mart tarafından desteklenmiştir. MACS teknolojisi, onun bakış açısı ile muazzam yardımcı Eleanor Brown (Miltenyi Biotec, Auburn, CA) için ayrılmış özel bir teşekkür ediyorum. Miltenyi cömertçe deneyler gösteri için hiçbir ücret ödemeden kullanılan malzeme sağladı. Ben de hücre hatları ifade PKCζ oluşturulan Dr. Guanghu Wang (Medical College of Georgia / Gürcistan Sağlık Bilimleri Üniversitesi, Augusta, GA) minnettarım. Destek Gürcistan / Gürcistan Sağlık Bilimleri Üniversitesi (Dr Lin Mei müdürlüğüne altında) Tıp College Moleküler Tıp Enstitüsü tarafından da kabul edilmektedir.

1. Giriş Manyetik aktive hücre sıralama (LIMACS) tekniği kullanılarak lipid vezikül aracılı afinite kromatografisi 1-3 seramid ile ilişkili protein kompleksleri izole etmek için laboratuvarda geliştirilmiştir. Başlangıçta, lipid veziküller manyetik partikül konjuge Annexin V (fosfatidilserin için son derece ilgin) kullanarak MACS veziküller ve ilişkili proteinler izole etmek için izin seramid ve fosfatidilserin yapılmıştır. Seramid ile ilişkili bir polarite karmaşık ve seramid-bağlayıcı proteinlerin hücre izolasyonu in vitro sulandırıldıktan 3 lizatları LIMACS tekniği kullanılmıştır. LIMACS veziküller (örneğin, glikolipid specifc lektinler veya lipid antikorlar) izolasyonu için diğer etkileşim ortaklarını kullanarak değiştirilebilir. 2. Deneysel Prosedürler Lipid Veziküller hazırlanması ve aPKCBinding Tahliller <ol><li> Lipid veziküller fosfatidilserin (105 mikrogram) ve büyük lipozom hazırlama 1,4-7 için modifiye işlemleri takiben C16-seramid (85 mikrogram) ekimolar miktarda kurutulmuş karışımlar elde edilir. </li><li> Lipid karışımları sonra yeniden süspanse ve 1 saat 50 mM Tris / HCl (pH 7.5) ve 150 mM NaCl içeren vezikül tampon 100 ul sonicated. </li><li> 0.1 mM MnCl 2 ile desteklenmiş vezikül tampon 300 ul ekledikten sonra, örnekleri, 4 20 dakika 12.000 μ g ° C santrifüj </li><li> Pelet (büyük lipid veziküller) 100 ul vezikül tampon yeniden bekletildi ve 37 az 1 saat süreyle Vybrant CM-DII 1 nmol ile inkübe ° C MACS ayrılıktan sonra vezikül fraksiyonu görselleştirmek. Vybrant CM-DII özellikle lipid membranlar içeren kırmızı bir floresan boya. </li><li> Deterjan ücretsiz cep lizat santrifüj membranöz enkaz kaldırma hipotonik tampon (proteaz ve fosfataz inhibitörleri ile 10 mM Tris / HCl (pH 7.0)) 300 ul sonication / hücre homojenizasyon hazırlanmıştır. 1 saat için 100.000 xg&#39;de santrifüj adım fosfatidilserin içeren endojen membran ile hücre lizat kirlenmesini önlemek için ilave edilmelidir. </li><li> Temizlenir lizat lipid vezikül süspansiyon eklenir ve karışım 4 ° C&#39;de 2 saat inkübe </li><li> Reaksiyon karışımı, 20x Annexin V bağlayıcı Annexin V konjuge manyetik boncuklar içeren bir çözüm tampon ve 50 ul 20 ul ile desteklenmiş 1 saat 4 ° C inkübasyon takip </li><li> MACS üreticinin (Miltenyi Biotec, Inc.) Protokolü göre yapılır. Mikroplaka floresan okuyucu kullanarak akış yoluyla ve elüsyon kesirler Vybrant CM-DII floresan izlenmesi lipid veziküllerin varlığı ve miktarı tarafından belirlenir. </li><li> Veziküler lipit miktarı ve floresan yoğunluğu arasında doğrusal bir ilişki vezikül bağlı Vybrant CM DII Annexin V-MACS uygulanan lipid karışımı kantitatif yüksek performanslı ince tabaka kromatografisi (HPTLC) tarafından doğrulanmadı. </li><li> Seramid / fosfatidilserin veziküller aPKC veya diğer proteinleri bağlama reaksiyon özgüllüğü 1 saat 4 ° C inkübasyon öncesinde hücre lizat inkübe 1 mikrogram anti-PKCζ tavşan poliklonal antikor kullanarak bir antikor yarışma yöntemi ile doğrulanır lipid veziküllerin. </li><li> MACS eluat seramid / fosfatidilserin veziküller bağlayıcı protein, SDS-PAGE ve immunoblotting tarafından analiz edilir. </li></ol> In vitro lipid-protein polarite kompleksi <ol><li> In vitro lipid-protein polarite kompleksi sulandırıldıktan, önceki bölümde açıklandığı gibi LIMACS prosedürü izleyerek yapılır . Kısa, fosfatidilserin (420 mikrogram) ve C16-seramid (107 mikrogram), organik çözücü kurutulur. </li><li> Kurutulmuş lipidler ul vezikül tampon 500 (150 mM NaCl, 50 mM Tris / HCl, pH 7.5) sonication altında yeniden süspanse. </li><li> 10 mM MnCl2, 1 ul Vybrant CM-DII ve PKCζ, 500 ng (insan rekombinant) Beş ul eklenir ve reaksiyon karışımı 4 undergentle ajitasyon 60 dakika inkübe edilir ° C </li><li> Vybrant CM-DII lekeli fosfatidilserin / seramid veziküller 60 dk 4 ° C 12.000 xg&#39;de santrifüj tarafından kurtarıldı </li><li> Pelet (pembe) 100 ul Tris tamponu yeniden süspanse ve γS-GTP (100 mcM), GSYH (1 mM), GST-Par6 (100 ng), ya da GST-Cdc42 (500 ng) ile desteklenmiş ve daha 3 inkübe 4 h ° C (ilgi rekombinant proteinlerin başka herhangi bir kombinasyonu kullanılabilir). </li><li> Annexin V-tampon (20x stok solüsyonu 5 ul) ve Annexin V-konjuge manyetik boncuk (50 ul) eklenir ve reaksiyon karışımı yavaşça sallanarak başka bir 30 dakika 4 ° C&#39;nin altında inkübe </li><li> Annexin V-MACS tedarikçinin protokolü daha önce açıklandığı gibi yapılır. </li&gt; <li> Elüsyon fraksiyonu (1 ml), 10 mikrogram ile yağış yardımcı olarak saf ovalbumin tamamlanmaktadır. Protein Wessel-Flugge yağış konsantre ve daha önce 8 açıklandığı gibi SDS-PAGE/immunoblotting tarafından analiz edilir. </li><li> Wessel Flugge yağış reaksiyon organik (kloroform / metanol) aşamasında Vybrant CM-DII (pembe) tespiti ile yıkandı, lipid veziküllerin miktar miktarı. Analiz protein miktarı eşit miktarda lipid veziküllerin normalize. </li></ol> 3. Sonuçlar LIMACS PKCζ-EGFP arıtma ve seramid bağlayıcı etki alanı C20ζ-EGFP Tam uzunlukta ifade MDCK hücreleri bir deterjan içermeyen lizat PKCζ C-terminal, yeşil floresan protein (FLζ EGFP) veya PKCζ (C20ζ-EGFP) C-terminalindeki bir seramid bağlayıcı etki alanı bağlantılı fosfatidilserin / seramid veziküller ile inkübe Deneysel Prosedürler nitelendirdi. MACS sütun elüsyon sonra, protein tespiti için yıkandı, protein 2 PKCζ ve EGFP immunoblotting ve karşı antikorlar kullanılarak analiz edildi. <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="Şekil 1" /> Şekil 1 LIMACS EGFP-etiketli PKCζ ve C-terminal parçası C20ζ fosfatidilserin / seramid veziküller. Deneysel Usul bölümünde açıklandığı gibi, EGFP (bağlayıcı olmayan bir kontrolü gibi), tam uzunlukta PKCζ-EGFP veya seramid bağlama, C-terminal parçası C20ζ-EGFP ifade MDCK hücreleri Deterjan lizatları fosfatidilserin / seramid veziküller ile inkübe edildi. . LIMACS kullanarak sonra, protein, SDS örnek tamponu ile yıkandı, ve SDS-PAGE ile analiz ve immunoblotting. Sol panelinde EGFP Annexin V bağlantılı manyetik boncuk muhafaza veziküller bağlamak olmadığını gösterir. Orta ve sağ panelde o tam uzunlukta PKCζ-EGFP C20ζ-EGFP seramid için bağlayıcı nedeniyle tutuldu.

<ol>
	<li>Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+to+ceramide+activates+protein+kinase+Czeta+before+the+formation+of+a+pro-apoptotic+complex+with+PAR-4+in+differentiating+stem+cells.">Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.</a> J Biol Chem. 280, 26415-26424 (2005).</li><li>Wang, G., Krishnamurthy, K., Umapathy, N. S., Verin, A. D., Bieberich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxyl-terminal+domain+of+atypical+protein+kinase+Czeta+binds+to+ceramide+and+regulates+junction+formation+in+epithelial+cells.">The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.</a> J Biol Chem. 284, 14469-14475 (2008).</li><li>Chalfant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+ceramide+regulates+the+alternative+splicing+of+caspase+9+and+Bcl-x+in+A549+lung+adenocarcinoma+cells.+Dependence+on+protein+phosphatase-1.">De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.</a> J Biol Chem. 277, 12587-12595 (2002).</li><li>Simon, C. G., Holloway, P. W., Gear, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+C(16)-ceramide+between+phospholipid+vesicles.">Exchange of C(16)-ceramide between phospholipid vesicles.</a> Biochemistry. 38, 14676-14682 (1999).</li><li>Goni, F. M., Contreras, F. X., Montes, L. R., Sot, J., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+(and+sociology)+of+ceramides.">Biophysics (and sociology) of ceramides.</a> Biochem Soc Symp. , 177-188 (2005).</li><li>Kumagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CERT+mediates+intermembrane+transfer+of+various+molecular+species+of+ceramides.">CERT mediates intermembrane transfer of various molecular species of ceramides.</a> J Biol Chem. 280, 6488-6495 (2005).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Anal Biochem. 138, 141-143 (1984).</li></ol>

Hücre zarında lipid gömerek bir lipid ve bağlayıcı protein arasında belirli bir etkileşim test etmek için bir engel teşkil etmektedir. Hücre zarı, birkaç lipitleri ve proteinleri karışımından oluşur ve lipid microdomains veya sallar düzenlenmiştir. Bu nedenle, bir protein, lipid doğrudan bağlar ya da sadece bir microdomain yapısı zenginleştirilmiş microdomains ve proteinlerin ortak arıtma açıkça ayırt edemez. Lipid ELISA&#39;larla veya Plasmon rezonans spektroskopi gibi yüzeyleri kaplanm?...

Annexin V-konjuge manyetik boncuk ve MACS mikro ve minicolumns Miltenyi Biotec, Inc (Auburn, CA) tarafından verilmiştir. Tüm lipidler yüksek saflıkta ve Avanti Polar lipidler (Alabaster, AL) elde edilen.

lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction

Lipidler çoğu arıtma prosedürleri erişilemez duruma, bu nedenle hücre zarlarında gömülü ve çünkü lipid protein etkileşim analizi zordur. Alternatif olarak, lipidler, lipid ELISA ve Plazmon rezonans spektroskopi için kullanılan gibi düz yüzeyler üzerinde kaplı olabilir. Ancak, yüzey kaplama lipidler, lipid bağlayıcı özellikleri için önemli olabilir microdomain yapılar oluşturmak yok. Ayrıca, bu yöntemleri, hedef lipidler bağlayıcı proteinlerin büyük miktarlarda arıtma için izin vermez. Lipid protein etkileşim test bu sınırlamaları aşmak ve lipid bağlayıcı proteinlerin arındırmak için manyetik aktive hücre sıralama (LIMACS) kullanarak lipid vezikül aracılı afinite kromatografisi olarak adlandırılan yeni bir yöntem geliştirdi. Bu yöntemde, lipid veziküller Annexin V MACS çapa lipid olarak hedef lipid ve fosfatidilserin ile hazırlanmıştır. Fosfatidilserin protein Annexin V. fosfatidilserin içeren lipid veziküller manyetik boncuk bağlamak Annexin V konjuge manyetik boncuklar kullanarak yüksek afinite gösteren her yerde bulunan bir hücre zarı fosfolipid. Hedef lipid bağlayıcı protein, lipid veziküller lizat bir hücre ile inkübe edilir boncuklar bağlı olacak ve MACS kullanarak ortak arındırılmalıdır. Bu yöntem ayrıca test etmek için kullanılır rekombinant proteinler sulandırmak hedef lipid bağlayıcı bir protein kompleksidir. Biz sphingolipid seramid ve co-purify prostat apoptozis cevabı 4 (PAR-4), seramid ilişkili aPKC için bağlayıcı bir protein atipik PKC (aPKC) etkileşimi göstermek için bu yöntemi kullanmıştır. Ayrıca hücre polarite ilgili proteinler Par6 ve Cdc42 rekombinant aPKC seramid ile ilişkili kompleks sulandırıldıktan için bu yöntemi kullanmıştır. Lipid veziküller sphingo-veya fosfolipitler çeşitli hazırlanmış olabilir, hücre zarından yakından benzer bir lipid ortamda LIMACS lipid-protein etkileşim için çok yönlü bir test sunuyor. Ek lipid protein kompleksleri lipid bağlayıcı protein, lipid veziküller ile birlikte arıtılmış proteomik analizi kullanılarak tespit edilebilir.

Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

MACS ve Annexin V-konjuge manyetik boncuk ve hedef lipid ve Annexin V-bağlayıcı fosfatidilserin sentezlenen lipid veziküller hedef lipid protein etkileşim test etmek için. Ortak hedef lipid bağlı Proteinler saflaştırılmış ve boncuk elüsyon sonra analiz edilir.

Manyetik Aktif Hücre Ayırma (LIMACS) kullanarak Lipid veziküllü aracılı Afinite Kromatografisi: Protein-lipit Etkileşim Analiz Roman Yöntemi

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

lipid-vesicle-mediated-affinity-chromatography-using-magnetic

Research

JoVE Journal

Biology

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

12.4K Görüntüleme.  Georgia Health Sciences University. MACS ve Annexin V-konjuge manyetik boncuk ve hedef lipid ve Annexin V-bağlayıcı fosfatidilserin sentezlenen lipid veziküller hedef lipid protein etkileşim test etmek için. Ortak hedef lipid bağlı Proteinler saflaştırılmış ve boncuk elüsyon sonra analiz edilir.

Video: Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Bitki protoplast Floresan Aktif Hücre Ayırma

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Biyoloji

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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 ...

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 ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Hücre Özgül Analizi<em&gt; Arabidopsis</em&gt; Floresans Aktif Hücre Ayırma kullanma Leaves

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Cep Takip Boyalar kullanarak Hücre Bölünmesi İzleme için optimize Boyama ve Çoğalma Modelleme Yöntemleri

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

Anne Hücreleri ve dalgalanma Testleri Manyetik orting birleştiren Mitotic Hücre Yaşlanma sırasında Genom istikrarsızlık Analiz için<em&gt; Saccharomyces cerevisiae</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Tandem Affinity Arıtma kullanarak MyoD interaktom belirlenmesi kütle spektrometresi ile birleştiğinde

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Manyetik aktif hücre stratejileri yalıtmak ve Sinovyal sıvı elde edilen mezenkimal kök hücre bir tavşan modelinden arındırmak için sıralama

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...