Diese Arbeit wurde durch Fördermittel der Deutschen Forschungsgemeinschaft GraKo1121, Hu1011/2-1 und SFB940 SH unterstützt

Hinweis: Detaillierte Informationen zur Puffer im Protokoll angegeben ist in Tabelle 1 verfügbar. 1. Fixierung von Protein-Protein-Interaktionen durch Formaldehyd-Vernetzungs in lebenden Zellen <ol><li> Um zu beginnen vorzubereiten Wachstumsmedien, Stammlösungen und Puffer P1 <ol><li> Bereiten 500 ml Medium: Das Medium sollte Bedingungen, die die Wechselwirkung zwischen Membran Köderprotein und Beuteproteine ​​unterstützen. Darüber hinaus sollte ein Versuch unter Bedingungen, bei denen keine Wechselwirkung zu erwarten ist (z. B. ohne Vernetzung) durchgeführt werden. Anmerkung: Wir vergleichen Protein-Protein-Wechselwirkungen bei gestressten und nicht gestressten Bakterien. Daher bereiten wir 500 ml Luria Bertani (LB)-Medium (Tabelle 1), die wir mit einem Stress-Induktionsmittel (zB 0,5 M NaCl) in einem 2 l-Erlenmeyerkolben zu ergänzen. Zur Steuerung verwenden wir normale LB-Medium mit 0,17 M NaCl (pH 7,0). </li><li> Vorbereitung Tris-Puffer (20 mM Tris-HCl, pH 8,0) und 0,1 M Ethylendiamintetraessigsäure (EDTA), pH-Wert 8,0-Stammlösung (Tabelle 1). </li><li> Bereiten Puffer P1. Auflösen Saccharose auf eine Endkonzentration von 0,5 M Tris-Puffer. Sterilisieren Puffer P1 durch Filtration und speichern sie bei 4 ° C </li></ol></li><li> Wachsen Zellen, die den Membranköderprotein mit einem C-terminalen Strep-tag-Fusions von einem Vektor exprimiert. Induzieren die Expression von Membranproteinen Köder für eine ausreichende Zeit. Anmerkung: Wir induzieren die Expression der bakteriellen Membran Köderproteine ​​in einem frühen logarithmischen Phase (A 600 = 0,3) durch Zugabe von 250 ul 1 M isopropyl-β-D-thiogalactopyranosid (IPTG) bis zu 500 ml Medium (Endkonzentration: 0,5 mM) . Um eine ausreichende Expression zu ermöglichen, Inkubation wir die Bakterien bis in die späten Log-Phase (A 600 = 1.2). </li><li> Bereiten Sie für jede Kultur zwei Zentrifugenbecher mit einem Mindestvolumen von jeweils 300 ml und legen sie auf Eis. </li><li> Führen Formaldehyd-Vernetzung. CAUTION: Formaldehyd ist giftig Wir empfehlen unter einem Sicherheits Abzug arbeiten.. <ol><li> Übertragen Sie das Kulturgefäß unter einer Abzugshaube Sicherheit. Aufgeteilt jede Probe in zwei Proben, eine Auslassung Vernetzungs (- X) und eine darunter Formaldehydvernetzungs (X +). 4 ml 37% ige Formaldehydlösung und 250 ml Kultur bis zu einer Endkonzentration von 0,6% zu erreichen. </li></ol></li><li> Übertragen Sie die Kulturgefäße zurück und wachsen Zellen für weitere 20 min wie zuvor. </li><li> Füllen Sie Ihre Kulturen in die Zentrifugenröhrchen unter einem Sicherheits Abzug. Sammle die Zellen durch Zentrifugation bei 3.000 × g für 30 min. </li><li> Überstand verwerfen durch vorsichtiges Pipettieren es unter einer Abzugshaube Sicherheit. Sammeln giftige Formaldehyd-haltigen Überstand und entsorgen Sie es ordnungsgemäß. </li><li> Um eine optimale Ausbeute bei der Membranproteinpräparat zu erreichen, bereiten erste Spheroplasten. Hier stellen wir ein Protokoll für Sphäroblastenbildung von Gram-negativen Bakterien: <ol><li> Prepare Puffer P2 (Tabelle 1) von lyophilisierten Pulvers. Leicht auflösen 2 mg Lysozym in 0,1 M EDTA, pH 8, in einem 1,5-ml-Röhrchen. Immer frisch zubereiten Puffer P2 für den Tag des Experiments. </li><li> Vorbereitung Tris-Puffer mit Protease-Inhibitor (Puffer P3). Zu 10 ml Tris-Puffer, 0,1 ml 1 M Phenylmethylsulfonylfluorid (PMSF) (Tabelle 1) bis zu einer Endkonzentration von 10 mM. Verwenden Sie die Puffer sofort, um die ordnungsgemäße Funktion der PMSF als Protease-Inhibitor zu gewährleisten. </li><li> Resuspendieren Zellpellets in 10 ml Tris-Puffer mit Protease-Inhibitor und Transferlösung in ein 15 ml konischen Röhrchen. </li><li> 1 ml Puffer P2 und Inkubation auf Eis für 30 min. </li><li> Sammeln Spheroplasten durch Zentrifugation bei 3000 × g für 30 min und Überstand verwerfen sorgfältig. </li></ol></li><li> Sphäroplast die Pellets über Nacht bei -20 ° C inkubieren </li></ol> 2. Reinigung von Strep-Tag Membrane Protein (Bait) <ol><li> Zeigen Sphäroplast Pellet auf Eis. &lt;/ Li&gt; <li> Während der Zeit, die Sphäroplast Pellets auf Eis auftauen, bereiten 10 ml Puffer P3 für jeden Spheroplastenpräparation. Vorsichtig lösen 1 mg DNaseI in 10 ml Tris-Puffer. 0,1 ml 1 M PMSF kurz vor der Verwendung. </li><li> Das Pellet in 6 ml frisch zubereitet P3. Beschallen die Probe viermal für 1 min kontinuierlich auf Eis mit 1 min Pause zwischen jedem Burst, um die Sphäroplasten zu stören. </li><li> Zentrifugieren Sie die Probe bei 10.000 g für 10 min bis zur Ernte Zelltrümmer. Den Überstand mit einer Pipette, ein Ultrazentrifugenröhrchen. </li><li> Pellet die Membranfraktion bei 100.000 × g für 30 min. In Tris-Puffer waschen das Pellet vorsichtig, ohne dass es sich auflöst. Trocknen Sie das Rohr mit einem Papiertuch (zB Kleenex). Vermeiden Sie die Pelletjederzeit zu stören. </li><li> Das Pellet (= Membranen) vorsichtig in 1 ml Tris-Puffer. Verwenden ein 20 ul Aliquot der Proteinkonzentration durch zB BCA-Assay zu bestimmen. Bei diesem Schritt können die Membranen Schock li gefrorenquid Stickstoff und bei -80 ° C gelagert </li></ol> 3. Reinigung von Strep-Tag Membrane Protein-Köder-und SDS-PAGE <ol><li> Normalisierung der Proteinkonzentration der Membranfraktion zu 5 mg / ml mit Tris-Puffer. Nehmen Sie 2.5 ml Membranfraktion (5 mg / ml) in ein Ultrazentrifugenröhrchen und Zugabe von 0,25 ml 20% Triton X-100 auf eine Endkonzentration von 2% zu erreichen, um die Membranproteine ​​zu solubilisieren. Fügen Sie eine Mikro-Magnetstab und rühren auf Eis für 1 Stunde. Anmerkung: Obwohl im Allgemeinen haben wir gute Ergebnisse mit Triton X-100 als Detergens, könnte es sinnvoll, die Reinigungsmittel für die Optimierung zu ändern. In dieser Hinsicht haben wir die besten Ergebnisse mit solchen Reinigungsmitteln für die Reinigung von funktionellen jeweiligen Membran Köderprotein verwendet. </li><li> Während der Ausführung von Schritt 3.1, bereiten 50 ml Puffer B und 5 ml von Puffer E (Tabelle 1). Füllen Sie 10 ml 5x Puffer W Konzentrat auf 50 ml und fügen Sie 150 ul 20% Triton X-100. <ol><li> Füllen Sie 1 ml 5x buffer E-Konzentrat auf 10 ml und fügen Sie 30 ul 20% Triton X-100. Ins Gleichgewicht einer 1 ml Strep-Tactin Superflow Schwerkraftsäule mit 8 ml Puffer W. Hinweis: Es ist wichtig, um das Reinigungsmittel zur Solubilisierung in einer Konzentration 10-fach über der kritischen Mizellenkonzentration (CMC) in jedem Puffer während des Reinigungsverfahrens verwendet hinzuzufügen. </li></ol></li><li> Entfernen der mikromagnetischen Stab aus der Solubilisierung Probe und Ultrazentrifuge bei 100.000 × g für 30 min, um das unlösliche Membranfraktion zu pelletieren. Entfernen Sie den Überstand mit einer Pipette zur weiteren Reinigung. </li><li> Laden der Überstand auf die Säule. Führen Sie die Spalte nur mit Schwerkraft. Die Säule wird mit 5 ml Puffer W. Wiederholen Sie diesen Waschschritt 5x. </li><li> Eluiere Membranproteine ​​Köder mit 1 ml Puffer E. Wiederholen Sie diesen Schritt 4x Elution. </li><li> Konzentrieren Elutionsfraktionen 2, 3, und 4 bis 300 ul einer Zentrifugalfiltereinheit. </li><li> Mischungs 200 &amp;mgr; l jeder Probe mit 50 &amp;mgr; l 5 × SDS-PAGE Ladefarbstoff. Split jedes Präparat in 125 ul Aliquots. Man kocht ein Aliquot von jedem Präparat 20 Minuten lang bei 95 ° C zu Formaldehyd vernetzt umkehren. Lassen Proben abkühlen auf RT für mindestens 10 Minuten auf der Bank der Spitze. </li><li> Last 30 ul von jeder Probe auf eine einzelne Spur einer Polyacrylamid-Gel für Immunoblotting. Verwenden Sie eine vorgefärbte Molekulargewichtsmarker für beste Orientierung und führen SDS-PAGE-10. </li></ol> 4. Immunoblot-Analyse, um Interaktionspartner bestätigen <ol><li> Sobald der Schritt 3.8 abgeschlossen ist, die Übertragung Proteine ​​auf eine Nitrocellulose-Membran durch halbtrockenen zB. </li><li> Blockieren der Membran, um unspezifische Markierung zu verhindern. Vorbereitung Blockierungspuffer durch Wiegen Rinderserumalbumin (BSA) zu 3% Gewicht pro Volumen in Tris-gepufferter Kochsalzlösung mit einer endgültigen Konzentration von 0,05% Tween-20 (TBS-T) ergänzt erzielen. Verwenden Sie eine ausreichende Menge an Blockierungspuffer, um die Membran zu decken. Sperrung der Membran für 1 h bei RT. </li><li> Verdünnen Sie einnd bebrüten die Beute Protein-spezifischen ersten Antikörper wie gewohnt. Verwenden ein HRP-verknüpften Antikörper als der zweite Antikörper. </li><li> Entwickeln Sie die Immunoblot mit einem Chemilumineszenz-Nachweis-Kit mit hoher Empfindlichkeit. Überwachung des Signals mit einem klassischen Film Veredelung oder Digital-Imaging-Ausrüstung. </li></ol> 5. NanoLC-ESI-MS/MS Hohe Auflösung Experimente zur Wechselwirkung Partner identifizieren <ol><li> Im Fall, dass keine spezifischen Antikörper für die Beuteprotein oder unbekannte Interaktionspartner verfügbar gemacht werden sollen, verwenden Sie die Massenspektrometrie (MS) zur Identifizierung. Um Keratin Kontamination zu verhindern, verwenden vorgefertigte Gele für die Protein-Trennung. </li><li> Silberfärbung der SDS-PAGE unter Verwendung eines MS-kompatibel Färbekit nach dem Protokoll des Herstellers. Führen Sie alle Färbe-und Waschschritte in Glastanks. </li><li> Verbrauch jeweiligen Bands und analysieren diese durch hochauflösende LC / MS-9. </li></ol>

<ol>
	<li>Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.</a> J. Mol. Biol. 305, 567-580 (2001).</li><li>Bakheet, T. M., Doig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+identification+of+human+protein+drug+targets.">Properties and identification of human protein drug targets.</a> Bioinformatics. 25, 451-457 (2009).</li><li>Yildirim, M. A., Goh, K. I., Cusick, M. E., Barabasi, A. L., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-target+network.">Drug-target network.</a> Nat. Biotechnol. 25, (2007).</li><li>Daley, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assembly+of+membrane+proteins+into+complexes.">The assembly of membrane proteins into complexes.</a> Curr. Opin. Struct. Biol. 18, 420-424 (2008).</li><li>Jung, K., Fried, L., Behr, S., Heermann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histidine+kinases+and+response+regulators+in+networks.">Histidine kinases and response regulators in networks.</a> Curr. Opin. Microbiol. 15, 118-124 (2012).</li><li>M&#252;ller, V. S., Jungblut, P. R., Meyer, T. F., Hunke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-SPINE:+An+improved+method+to+identify+protein-protein+interaction+partners+of+membrane+proteins+in+vivo.">Membrane-SPINE: An improved method to identify protein-protein interaction partners of membrane proteins in vivo.</a> Proteomics. 11, 2124-2128 (2011).</li><li>Herzberg, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPINE:+A+method+for+the+rapid+detection+and+analysis+of+protein-protein+interactions+in+vivo.">SPINE: A method for the rapid detection and analysis of protein-protein interactions in vivo.</a> Proteomics. 7, 4032-4035 (2007).</li><li>Sutherland, B. W., Toews, J., Kast, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+formaldehyde+cross-linking+and+mass+spectrometry+in+the+study+of+protein-protein+interactions.">Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions.</a> J. Mass Spectrom. 43, 699-715 (2008).</li><li>Hernandez, P., M&#252;ller, M., Appel, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+protein+identification+by+tandem+mass+spectrometry:+Issues+and+strategies.">Automated protein identification by tandem mass spectrometry: Issues and strategies.</a> Mass Spectrom. Rev. 25, 235-254 (2006).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Hunke, S., Keller, R., M&#252;ller, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+by+the+Cpx-envelope+stress+system.">Signal integration by the Cpx-envelope stress system.</a> FEMS Microbiol. Lett. 326, 12-22 (2012).</li><li>Weber, R. F., Silverman, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cpx+proteins+of+Escherichia+coli+K12+:+Structure+of+the+CpxA+polypeptide+as+an+inner+membrane+component.">The Cpx proteins of Escherichia coli K12 : Structure of the CpxA polypeptide as an inner membrane component.</a> J. Mol. Biol. 203, 467-478 (1988).</li></ol>

Wir haben nichts zu offenbaren.

Membran SPINE Analyse ist ein biochemischer Ansatz, ermöglicht ein, um zu bestätigen und um zu diesem Punkt unbekannte Interaktionspartner von Membranproteinen zu identifizieren. Membran SPINE vereint in vivo Vernetzung durch Formaldehyd mit Reinigung eines Strep-markierten Membranköderprotein. Die Kombination mit Immunoblotting erleichtert den Nachweis von vorhergesagten Wechselwirkungspartner (Fig. 2). Zusätzlich wird die Verbindung mit MS-Analyse erlaubt die Identifikation unbekannter In...

Um die Funktion eines Proteins zu verstehen, ist es wichtig, seinen Interaktionspartnern kennen. Mehreren klassischen Techniken zur Identifizierung von Interaktionspartnern von löslichen Proteinen zur Verfügung. Jedoch sind diese Techniken nicht ohne weiteres übertragbar, um Proteine ​​aufgrund ihrer hydrophoben Natur 4 Membran. Um diese Einschränkung zu überwinden, haben wir die Membran Strep-Protein-Wechselwirkung Experiment (Membran-spine) 6 entwickelt. Es basiert auf der Methode DORN, die nur für lösliche Proteine ​​geeignet war 7 basiert. Membran-SPINE profitiert von zwei Vorteile des Vernetzungsmittels Formaldehyd: erste Formaldehyd kann leicht durchdringen Membranen und erzeugt eine genaue Momentaufnahme der Interaktom einer lebenden Zelle 8 daher. Zweitens kann Formaldehyd vernetzt durch Kochen 9 umgekehrt werden. Hier werden diese beiden Vorteile verwendet, um nicht nur dauerhaft, sondern auch vorübergehende PPI Membran Prote identifizierenIns 6. Kurz gesagt, ein Strep-Tag an den C-Terminus des integralen Membranköderprotein fusioniert ist. Zellen, die den Membranköderprotein exprimieren, werden mit Formaldehyd vernetzt Beute-Proteine ​​an die Membran Köder-Protein (Fig. 1) inkubiert. Modifikationen der Beuteproteine ​​sind nicht erforderlich. Als nächstes wird die Membranfraktion bereit. Daher sind Membranproteine ​​durch Detergens-Behandlung und Köder-Proteine ​​sind mit ihrer Beute Proteinen unter Verwendung von Affinitätsreinigung gereinigt Zusammenarbeit gelöst. Anschließend wird die Vernetzung durch Kochen umgekehrt, und der Köder und deren koeluierte Beute-Proteine ​​werden durch SDS-PAGE getrennt. Schließlich kann Beuteproteine ​​durch Immunoblot-Analyse bzw. Massenspektroskopie identifiziert werden.

<table border="0" cellpadding="0" cellspacing="0" width="878">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">37% Formaldehyde</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">4979.1</td>
			<td style="width:271px;">Should not be older than one year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DNaseI</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:137px;">DN25</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BCA protein assay kit</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">23225</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Micromagnetic rod</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">0955.2</td>
			<td style="width:271px;">5 mm in length, 2 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Triton X-100</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">6683.1</td>
			<td style="width:271px;">standard detergent for solubilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">n-Dodecyl-&#946;-maltoside</td>
			<td style="width:128px;">Glycon</td>
			<td style="width:137px;">D97002-C</td>
			<td style="width:271px;">the best detergent to solubilze CpxA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">1 ml Strep-Tactin superflow gravity flow column</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1207-050</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Strep-tag protein purification buffer set</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1002-001</td>
			<td style="width:271px;">Contains buffer W and buffer E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Amicon Ultra-4 centrifugal filter</td>
			<td style="width:128px;">Millipore</td>
			<td style="width:137px;">UFC803024</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">SuperSignal West Pico Chemiluminescent substrate</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">34080</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SilverQuest Silverstaining kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:137px;">LC6070</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FireSilver Staining Kit</td>
			<td style="width:128px;">Proteome Factory</td>
			<td style="width:137px;">P-S-2001</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ultracentrifuge</td>
			<td style="width:128px;"></td>
			<td style="width:137px;"></td>
			<td style="width:271px;"></td>
		</tr>
	</tbody>
</table>

membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

Membranproteine ​​sind essentiell für die Lebensfähigkeit der Zellen und sind daher wichtige therapeutische Ziele 3.1. Da sie in den Komplexen 4 funktionieren, sind Methoden zur Identifizierung und Charakterisierung ihrer Wechselwirkungen notwendig 5. Zu diesem Zweck haben wir die Membran Strep-Protein-Wechselwirkung Experiment genannt Membran-Dorn 6. Diese Technik kombiniert in vivo Vernetzen unter Verwendung des reversiblen Vernetzungsmittel Formaldehyd mit Affinitätsreinigung von Strep-tag eine Membran Köderprotein. Während des Verfahrens sind vernetzte Beuteproteine ​​mit der Membran Köderprotein co-gereinigt und anschließend durch Kochen getrennt. Daher können zwei wichtige Aufgaben bei der Analyse von Protein-Protein-Interaktionen (PPI) von Membranproteinen mit Membran-Dorn ausgeführt werden: erstens die Bestätigung einer vorgeschlagenen Interaktionspartner durch Immunoblotting, und zweitens die Identifizierung neuer Interaktionspartner von Massenspektrometrie-Analyse . Außerdem, selbst low Affinität, sind vorübergehende PPI durch diese Technik nachweisbar. Schließlich ist Membran-SPINE anpassungsfähig an nahezu jeden Zelltyp, so dass es zutreffend als ein leistungsfähiges Werkzeug, um Screening PPI von Membranproteinen zu identifizieren.

Membran SPINE-Analyse erlaubt die Co-Reinigung von Membranproteinen und transient interacting protein Partnern. Die Co-Reinigung unter Verwendung der Vernetzungsmittel Formaldehyd erreicht. Zwei Parameter sind wichtig, um unspezifische Vernetzungen verhindern: die Formaldehyd-Konzentration und der Vernetzungszeit. Die ausreichende, aber nicht übermäßige Verwendung von Formaldehyd kann leicht durch Immunoblotting gesteuert werden. Formaldehyd vernetztem Protein-Komplexe durch Kochen aber nicht durch SDS-Behandlung abg...

Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Eine biochemische Ansatz beschrieben, um die in vivo-Protein-Protein-Interaktionen (PPI) der Membranproteine ​​zu identifizieren. Das Verfahren kombiniert Proteinvernetzung, Affinitätsreinigung und Massenspektrometrie, und ist an fast jedem Zelltyp oder Organismus. Mit diesem Ansatz wird auch die Identifizierung der transienten PPI möglich.

Membran-DORN: Ein Tool, um biochemische Protein-Protein-Interaktionen von Membranproteinen Identifizieren<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

13.6K Aufrufe.  Universität Osnabrück.  Eine biochemische Ansatz beschrieben, um die in vivo-Protein-Protein-Interaktionen (PPI) der Membranproteine ​​zu identifizieren. Das Verfahren kombiniert Proteinvernetzung, Affinitätsreinigung und Massenspektrometrie, und ist an fast jedem Zelltyp oder Organismus. Mit diesem Ansatz wird auch die Identifizierung der transienten PPI möglich. 

Serie: Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Herstellung von elektrochemischen DNA Biosensoren für die Reagenzienfreier Nachweis von Nukleinsäuren, Proteinen und kleinen Molekülen

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Forschung

Biotechnik

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Visualizing Proteine ​​und Makromolekulare Komplexe von Negative Stain EM: von Grid Vorbereitung auf Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Entwurf eines zyklischen Druck Bioreaktor für die Ex Vivo Studium der Aorten-Herzklappen

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Bestimmung der Transport von Xenobiotika und Nanomaterialien über die Plazenta mit dem<em&gt; Ex vivo</em&gt; Menschliche Plazentaperfusion Modell

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Isolation und Charakterisierung von chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lumineszenz-Resonanzenergietransfer zu Konformationsänderungen in Membranproteinen in Säugerzellen exprimiert Studieren

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluoreszenz-Abschrecken eines Liposomenverkapseltes NIR-Fluorophore als Instrument zur<em&gt; In-vivo-</em&gt; Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Sandwichartigen Mikroumgebungen zu nutzen Handy / Material-Interaktionen

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Herstellung von 3D-Kollagen-Gel und Mikrokanäle für das Studium der 3D-Interaktionen<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA-Nanoröhren als ein vielseitiges Werkzeug, semiflexiblen Polymere zu studieren

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...