Diese Arbeit wurde durch die NIH gewährt R01NS046835 und R01AG034389 und der March of Dimes gewähren 6FY08-322 unterstützt. Ein besonderer Dank an Frau Eleanor Brown (Miltenyi Biotec, Auburn, CA), die enorm mit ihren Einsichten halfen in die MACS-Technologie gewidmet ist. Miltenyi hat großzügig das Material für die Demonstration der Experimente ohne Kosten verwendet werden, vorausgesetzt. Ich bin auch dankbar, dass Dr. Guanghu Wang (Medical College of Georgia / Georgia Health Sciences University, Augusta, GA), die PKCζ exprimierende Zelllinien generiert. Unterstützung durch das Institut für Molekulare Medizin an der Medical College of Georgia / Georgia Health Sciences University (unter Leitung von Dr. Lin Mei) ist ebenfalls anerkannt.

1. Einführung Die Lipid-Vesikel-vermittelten Affinitätschromatographie unter Verwendung magnetischer Zellsortierung (LIMACS)-Technik wurde in unserem Labor entwickelt zu isolieren Ceramid-associated Protein-Komplexe 1-3. Ursprünglich waren die Lipidvesikel von Ceramid und Phosphatidylserin, die für MACS mit magnetischen Partikel-konjugierten Annexin V (hoch affine zu Phosphatidylserin) zu den Vesikeln und deren assoziierten Proteinen zu isolieren erlaubt hat. Wir haben die LIMACS verwendete Technik für die in vitro Rekonstitution eines Ceramid-assoziierten Polarität komplexer und die Isolierung von Ceramid-bindenden Proteinen aus Zelllysaten 3. LIMACS modifiziert mit anderen Interaktionspartner für die Isolierung der Vesikel (zB Glycolipid-specifc Lektine oder Lipid-Antikörper) sein. 2. Experimentelle Verfahren Herstellung von Lipid-Vesikel und aPKCBinding Assays <ol><li> Lipidvesikel sind aus getrockneten Mischungen von äquimolaren Mengen von Phosphatidylserin (105 ug) und C16-Ceramid (85 ug) folgende modifizierte Verfahren für große Liposomenzubereitung 1,4-7 erhalten. </li><li> Die Lipid-Gemische werden dann resuspendiert und beschallt für 1 h in 100 ul Vesikel Puffer bestehend aus 50 mM Tris / HCl (pH 7,5) und 150 mM NaCl. </li><li> Nach Zugabe von 300 ul Vesikel-Puffer mit 0,1 mM MnCl 2 ergänzt, werden die Proben bei 12.000 μ g für 20 min bei 4 ° C zentrifugiert </li><li> Das Pellet (große Lipidvesikel) ist in 100 ul Vesikel-Puffer resuspendiert und mit 1 nmol Vybrant CM-DII für 1 h bei 37 ° C, um die Vesikelfraktion nach MACS Trennung zu visualisieren. Vybrant CM-DII ist ein roter Fluoreszenzfarbstoff speziell zur Einbindung in Lipidmembranen. </li><li> Ein Waschmittel-free Zelllysat wird durch Beschallung / Homogenisierung von Zellen in 300 ul hypotonischen Puffer (10 mM Tris / HCl (pH 7,0) mit Protease-und Phosphatase-Inhibitoren) durch Entfernung des membranösen Schutt, gefolgt von Zentrifugation vorbereitet. Ein Zentrifugationsschritt bei 100.000 xg für 1 h sollte hinzugefügt werden, um eine Verunreinigung des Zelllysat mit endogenen Membranen, die Phosphatidylserin zu vermeiden. </li><li> Das geklärte Lysat wird auf die Lipid-Vesikel Suspension zugegeben und das Gemisch wird für 2 h bei 4 ° C inkubiert </li><li> Das Reaktionsgemisch wird mit 20 ul 20x Annexin V-Bindungspuffer und 50 ul einer Lösung mit magnetischen Beads konjugiert Annexin V ergänzt, gefolgt von Inkubation für 1 h bei 4 ° C. </li><li> MACS ist nach Angaben des Herstellers (Miltenyi Biotec, Inc.) Protokoll durchgeführt. Das Vorhandensein und die Menge der Lipid-Vesikel wird durch die Überwachung der Vybrant CM-DII Fluoreszenz in den Flow-Through-und Elutionsfraktionen mit einem Mikrotiterplatten-Fluoreszenz-Reader bestimmt. </li><li> Die lineare Korrelation zwischen der Höhe der vesikulären Lipid-und Fluoreszenz-Intensität der Vesikel-gebundenem Vybrant CM-DII wird durch quantitative Hochleistungs-Dünnschichtchromatographie (HPTLC) der Lipid-Gemisch aufgetragen, um Annexin V-basierten Macs überprüft. </li><li> Die Spezifität der Bindungsreaktion aPKC oder andere Proteine, die Ceramid / Phosphatidylserin Vesikel wird durch einen Antikörper Kompetitionsassay mit 1 ug anti-PKCζ polyklonalen Kaninchen-Antikörpers gegen das Zelllysat für 1 h bei 4 ° C inkubieren vor der Inkubation mit verifizierten die Lipid-Vesikel. </li><li> Die Proteinbindung an Ceramid / Phosphatidylserin Vesikel in der MACS Eluat wird durch SDS-PAGE und Immunoblot analysiert. </li></ol> In-vitro-Lipid-Protein-Komplex Polarität <ol><li> Die in vitro Rekonstitution eines Lipid-Protein-Polarität Komplex ist nach dem LIMACS Verfahren wie im vorherigen Abschnitt beschrieben, durchgeführt. In kurzen, Phosphatidylserin (420 ug) und C16-Ceramid (107 ug) wird aus organischen Lösungsmittel getrocknet. </li><li> Die getrockneten Lipide sind unter Beschallung in 500 ul der Vesikel-Puffer (; 150 mM NaCl 50 mM Tris / HCl, pH 7,5) resuspendiert. </li><li> Fünf ul 10 mM MnCl 2, 1 ul Vybrant CM-DII, und 500 ng PKCζ (humaner rekombinanter) wird zugegeben und das Reaktionsgemisch inkubiert undergentle Agitation für 60 min bei 4 ° C. </li><li> Vybrant CM-DII gebeizt Phosphatidylserin / Ceramid Vesikel werden durch Zentrifugation bei 12.000 xg für 60 min bei 4 ° C wieder </li><li> Das Pellet (pink) wird in 100 ul Tris-Puffer und ergänzt mit γS-GTP (100 uM), BIP (1 mM), GST-Par6 (100 ng), oder GST-Cdc42 (500 ng) und weitere 3 inkubiert h bei 4 ° C (jede andere Kombination von rekombinanter Proteine ​​von Interesse kann hier eingesetzt werden). </li><li> Annexin V-Puffer (5 ul einer 20x Stammlösung) und Annexin V-konjugierten magnetischen Beads (50 ul) zugegeben und die Reaktionsmischung unter leichtem Schütteln für weitere 30 min bei 4 ° C inkubiert </li><li> Annexin V-MACS ist im Anschluss an die Lieferanten-Protokoll wie oben beschrieben durchgeführt. </li&gt; <li> Die Elutionsfraktion (1 ml) wird mit 10 pg des reinen Ovalbumin als Niederschlag Hilfe ergänzt. Das Protein wird durch Wessel-Flügge Fällung konzentriert und analysiert SDS-PAGE/immunoblotting wie zuvor 8 beschrieben. </li><li> Die Menge der eluierten Lipidvesikel wird durch den Nachweis von Vybrant CM-DII (rosa) in der organischen (Chloroform / Methanol) Phase des Wessel Flügge Fällungsreaktion quantifiziert. Die Menge an Protein analysiert wird auf gleiche Mengen von Lipidvesikeln normalisiert. </li></ol> 3. Ergebnisse LIMACS Reinigung von PKCζ-EGFP und die Ceramid-bindende Domäne C20ζ-EGFP Ein Waschmittel-free Lysat von MDCK-Zellen, die in voller Länge PKCζ C-terminal in Verbindung mit grün fluoreszierende Protein (FLζ-EGFP) oder Ceramid-bindende Domäne in der C-Terminus von PKCζ (C20ζ-EGFP) wurde mit Phosphatidylserin / Ceramid Vesikeln inkubiert beschrieben in experimentelle Verfahren. Nach der Elution der MACS-Säule wurde Protein analysiert mittels Immunoblot und Antikörper gegen PKCζ und EGFP für die Detektion der eluierten Protein 2. <img src="/files/ftp_upload/2657/2657fig1.jpg" alt="Abbildung 1" /> Abbildung 1. LIMACS von EGFP-markierten PKCζ und seine C-terminale Fragment C20ζ mit Phosphatidylserin / Ceramid Vesikel. Waschmittel-free Lysaten von MDCK-Zellen, die EGFP (als eine unverbindliche Regelung), in voller Länge PKCζ-EGFP oder der Ceramid Bindung, C-terminale Fragment C20ζ-EGFP wurden mit Phosphatidylserin / Ceramid Vesikeln inkubiert, wie in den experimentellen Verfahren beschrieben . Nach der Verwendung LIMACS, wurde Protein mit SDS-Probenpuffer eluiert und durch SDS-PAGE und Immunoblot. Die linke Tafel zeigt, dass EGFP nicht auf die Vesikel mit der Annexin V-linked magnetischen Kügelchen zurückgehalten zu binden. Die mittlere und rechte Bild zeigt die volle Länge PKCζ-EGFP und C20ζ-EGFP wurden durch die Bindung an Ceramid beibehalten.

<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>

Um zu testen, die spezifische Interaktion zwischen einem Lipid-und Protein wird durch die Einbettung von Lipiden in der Zellmembran behindert. Die Zellmembran besteht aus einer Mischung von mehreren Lipiden und Proteinen, und es ist in Lipid-Mikrodomänen oder Flöße organisiert. Daher kann die Co-Reinigung von Mikrodomänen und Proteine ​​nicht klar unterscheiden, ob ein Protein direkt bindet an ein Lipid oder nur in einem Mikrodomänenstruktur bereichert. Andere Methoden anhand definierter Lipide auf Oberflächen...

Annexin V-konjugierten magnetischen Beads und MACS Mikro-und Minisäulen wurden von Miltenyi Biotec, Inc. (Auburn, CA) zur Verfügung gestellt. Alle Lipide wurden von höchster Reinheit und erhielt von Avanti Polar Lipids (Alabaster, AL).

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

Die Analyse der Lipid-Protein-Wechselwirkung ist schwierig, da Lipide in Zellmembranen eingebettet sind und somit nicht zugänglich sind, die meisten Reinigungsverfahren. Als Alternative können Lipide auf flachen Oberflächen beschichtet werden, wie für die Lipid-ELISA und Plasmon-Resonanz-Spektroskopie eingesetzt. Allerdings tun Oberflächenbeschichtung Lipide bilden keine Mikrodomänenstruktur Strukturen, die möglicherweise für die Lipid-bindenden Eigenschaften wichtig. Darüber hinaus sind diese Methoden nicht für die Reinigung von größeren Mengen an Proteinen Bindung an ihr Ziel Lipide ermöglichen. Zur Überwindung dieser Grenzen testen Lipid-Protein-Interaktions-und Lipid-bindende Proteine ​​zu reinigen entwickelten wir eine neuartige Methode bezeichnet Lipidvesikel-vermittelte Affinitätschromatographie unter Verwendung magnetischer Zellsortierung (LIMACS). In diesem Verfahren werden Lipidvesikel mit dem Ziel Lipid Phosphatidylserin und als Anker für die Lipid-Annexin V MACS vorbereitet. Phosphatidylserin ist ein allgegenwärtiges Zellmembran Phospholipid, die eine hohe Affinität zum Protein Annexin V. Mit Magnetic Beads konjugiert Annexin V die Phosphatidylserin-enthaltende Lipid-Vesikel werden die magnetischen Kügelchen binden zeigt. Wenn die Lipid-Vesikel mit einer Zelle inkubiert Lysat die Proteinbindung an die Ziel-Lipid wird auch auf die Kügelchen gebunden und kann co-gereinigt werden mittels MACS. Diese Methode kann auch verwendet werden, um zu testen, ob rekombinanten Proteinen rekonstituieren einen Protein-Komplex Bindung an die Ziel-Lipid. Wir haben diese Methode benutzt, um die Interaktion von atypischen PKC (aPKC) mit den Sphingolipid Ceramid und die Zusammenarbeit zu reinigen Prostata Apoptose Antwort 4 (PAR-4), ein Protein-Bindung an Ceramid-assoziierten aPKC zeigen. Wir haben auch diese Methode für die Wiederherstellung eines Ceramid-assoziierten Komplex von rekombinanten aPKC mit der Zellpolarität-verwandte Proteine ​​Par6 und Cdc42 verwendet. Da Lipidvesikel mit einer Vielzahl von Sphingo-oder Phospholipiden hergestellt werden können, bietet LIMACS eine vielseitige Test für die Lipid-Protein-Wechselwirkung in einer Lipid-Umgebung, die sehr ähnlich ist, dass der Zellmembran. Weitere Lipid-Protein-Komplexe können unter Verwendung Proteomanalyse von Lipid-bindenden Proteins mit der Lipid-Vesikel co-gereinigt werden.

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

Um die Wechselwirkung eines Proteins mit seiner Ziel-Lipid verwendeten wir MACS und Annexin V-konjugierten magnetischen Kügelchen und Lipid-Vesikel, die aus der Ziel-Lipid-und Annexin V-Bindung Phosphatidylserin synthetisiert testen. Gebundene Proteine ​​zum Ziel Lipid sind co-gereinigt und analysiert nach der Elution von den Beads.

Lipidvesikel-vermittelte Affinitätschromatographie unter Verwendung von Magnetic Activated Cell Sorting (LIMACS): Eine neue Methode zur Protein-Lipid-Wechselwirkungen Analyze

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 Aufrufe.  Georgia Health Sciences University. Um die Wechselwirkung eines Proteins mit seiner Ziel-Lipid verwendeten wir MACS und Annexin V-konjugierten magnetischen Kügelchen und Lipid-Vesikel, die aus der Ziel-Lipid-und Annexin V-Bindung Phosphatidylserin synthetisiert testen. Gebundene Proteine ​​zum Ziel Lipid sind co-gereinigt und analysiert nach der Elution von den Beads.

Serie: 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.

Fluorescence Activated Cell Sorting von Pflanzenprotoplasten

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

Forschung

Biologie

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.

Isolierung von Labile Multi-Protein-Komplexe durch In vivo Kontrollierte Cellular Cross-Linking und Immuno-magnetische Affinitätschromatographie

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

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.

Avidität-basierte extrazelluläre Wechselwirkung Screening (AVEXIS) für die Erkennung von Scalable Low-Affinität extrazellulären Rezeptor-Ligand-Wechselwirkungen

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.

Eine flüssige Phase Affinity Capture Assay Verwendung von magnetischen Kügelchen, um Protein-Protein Interaktion Studie: Das Poliovirus-Nanobody Beispiel

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.

Zellspezifische Analyse<em&gt; Arabidopsis</em&gt; Blätter mit 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 ...

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.

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit 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 ...

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.

Die Kombination von Magnet Sortierung der Mutterzellen und Fluktuation Tests zu Genominstabilität Während mitotischen Zellalterung in Analysieren<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 ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Reinigung von nativem Komplexe für die Strukturstudie ein Tandem Affinity Tag-Methode unter Einsatz

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

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.

Magnetische aktiviert Zelle Sortieren Strategien zu isolieren und reinigen Synovial Flüssigkeit abgeleitet mesenchymalen Stammzellen aus einem Kaninchen-Modell

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

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Neuartige Methode der Transport der Plasmid-DNA zu Maus Blase Urothel durch Elektroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...