AF wurde durch einen Starting Grant des Europäischen Forschungsrates des Siebten Rahmenprogramms der Europäischen Gemeinschaft unterstützt (FP7 / 2007-2013) / ERC Finanzhilfevereinbarung Nr 203.413 und von der Minerva-Zentrum für Bio-Hybridkomplex systems.HA und AI werden unterstützt durch das Dalia und Dan Maydan Fellowship für fortgeschrittene Studierende an der Hebräischen Universität von Jerusalem.

1. Entwerfen einer Peptidarray <ol><li> Teilen die Sequenz des Zielproteins in teilweise überlappenden Peptiden 10-20 Resten. Variieren Sie die Größe der Überlappung von der spezifischen Experiment und die Ressourcen der Durchführung Labor, aber im Prinzip, je länger die Überlappung, desto besser. Bei der Gestaltung der Peptide Berücksichtigung bekannter Sekundärstrukturelemente in das Protein, das für die Interaktion verantwortlich sein kann dauern. </li><li> Bestellen Sie die entworfen Peptidarray durch kommerzielle Anbieter. Hierbei kann die Verwendung Peptide kovalent an die Anordnung durch den C-Terminus gebunden ist. </li></ol> 2. Blockieren nicht-spezifischer Bindung <ol><li> Einen 50 mM Tris oder Phosphat-Pufferlösung, enthaltend 0,05% Tween 20 (TBST / PBST) und Anpassung an den gewünschten pH-Wert mit einer abgemessenen Menge von HCl oder NaOH (um die exakte Ionenstärke weiß) und die gewünschte Ionenstärke mit NaCl . Hier verwenden einen pH-Wert von 7,5 und eine Ionenstärke von 150 mM. </li><li> Machen Sie eine Blockierungslösung von2,5% (w / v) Magermilchpulver in TBST / PBST. </li><li> Um unspezifische Bindung zu vermeiden, tauchen Sie das Array in 5 ml Blockierungslösung. Inkubieren des Arrays für 2-4 h bei Raumtemperatur oder über Nacht bei 4 ºC auf einem Schüttler. </li></ol> 3. Inkubation mit dem Protein <ol><li> Zuerst mit 5 ml Blockierungslösung für 30 Sekunden und dann zweimal mit 5 ml TBST / PBST für 5 min auf einem Schüttler bei Raumtemperatur Waschen des Arrays. </li><li> Inkubieren der gewaschenen Array mit 5 ml His-tagged Protein-Lösung, die 2,5% (w / v) Magermilchpulver zu verhindern, nicht-spezifische Bindung. Hier verwenden 4,5 &amp; mgr; M-Protein-Lösung (STIL 500-650) in der beschriebenen Blockierungslösung gelöst. HINWEIS: In der Regel 5-10 &amp; mgr; M-Protein für das Screening verwendet werden, aber die Proteinkonzentration kann sogar so niedrig wie 2-3 &amp; mgr; M, abhängig von der Bindungsaffinität und die lokalen Konzentrationen der Peptide, die auf dem Array gebunden (Effizienz der Synthese). Der Puffer, in dem das Proteinaufgelöst wird, kann variieren, ist aber üblich, die Protein verdünnt unter Verwendung des gleichen oben beschriebenen Blockierungslösung. </li><li> Inkubieren der Anordnung in der Proteinlösung für 3-8 h bei Raumtemperatur oder über Nacht bei 4 ºC. </li></ol> 4. Inkubation mit dem Antikörper <ol><li> Dreimal mit 5 ml TBST / PBST waschen Array. Die erste Wäsche wird für 30 sec, gefolgt von zwei Wäschen 5 min am Schüttler bei Raumtemperatur. </li><li> Inkubieren der gewaschenen Array mit 5 ml der verdünnten HRP-konjugiertem Antikörper (1: 1500) in einem Inkubationspuffer, der die gleichen Bestandteile enthält, in den gleichen Konzentrationen als Blockierungslösung für 1 Stunde auf einem Schüttler bei Raumtemperatur. Der Antikörper kann entweder das Zielprotein oder den Tag bindet. </li><li> Kontrollexperimente durchzuführen Testen der Wechselwirkung des Antikörpers mit Peptiden, die auf der Anordnung, insbesondere dann, wenn das Array Peptide von dem Zielprotein (zB zur Oligomerisierung Studien) durch Wiederholung des gleichen Protokoll ohne step 3, Inkubation mit dem Protein. </li><li> Dreimal mit 5 ml TBST / PBST waschen Array. Die erste Wäsche wird für 30 sec, gefolgt von zwei Wäschen 5 min am Schüttler bei Raumtemperatur. </li></ol> 5. Lesen Sie den Array <ol><li> Führen Sie Chemilumineszenz Entwicklung mit einer ECL-Western-Blot-Substrat-Kit. </li><li> Führen Sie die Erkennung mit einem Leucht Bildanalysators. </li><li> Analysieren Sie die Ergebnisse. Stellen Sie sicher, dass beide Duplikate auf dem Array zeigen die gleichen Signale, so dass die Ergebnisse zuverlässig sind. Wenn die Struktur der Proteinpartner bekannt ist, suchen auf der Struktur um die Bindungspeptide und nach den Bindungsstellen. Auch Peptide, die weit in der Sequenz sind nahe beieinander in der Tertiärstruktur und schaffen eine Bindungsstelle. </li></ol>

<ol>
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B., Buus, S., Schafer-Nielsen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Mapping+of+Linear+Antibody+Epitopes+in+Human+Serum+Albumin+Using+High-Density+Peptide+Arrays.">Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays.</a> PLoS ONE. 8 (7), (2013).</li><li>Uecker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+substrate+peptide+for+the+FLT3+receptor+tyrosine+kinase.">A substrate peptide for the FLT3 receptor tyrosine kinase.</a> British Journal of Haematology. 144 (1), 127-130 (2009).</li><li>Gabizon, R., Faust, O., Benyamini, H., Nir, S., Loyter, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relationship+studies+using+peptide+arrays:+the+example+of+HIV-1+Rev-integrase+interaction.">Structure-activity relationship studies using peptide arrays: the example of HIV-1 Rev-integrase interaction.</a> Med. Chem. Commun. 4 (1), 252-259 (2013).</li><li>Frank, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SPOT-synthesis+technique:+Synthetic+peptide+arrays+on+membrane+supports&#8212;principles+and+applications.">The SPOT-synthesis technique: Synthetic peptide arrays on membrane supports&#8212;principles and applications.</a> Journal of Immunological Methods. 267 (1), 13-26 (2002).</li><li>Falsey, J. R., Renil, M., Park, S., Li, S., Lam, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+and+Small+Molecule+Microarray+for+High+Throughput+Cell+Adhesion+and+Functional+Assays.">Peptide and Small Molecule Microarray for High Throughput Cell Adhesion and Functional Assays.</a> Bioconjugate Chemistry. 12 (3), 346-353 (2001).</li><li>Castiel, A., Danieli, M. 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+Stil+protein+regulates+centrosome+integrity+and+mitosis+through+suppression+of+Chfr.">The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr.</a> Journal of Cell Science. 124 (4), 532-539 (2011).</li><li>Amartely, H., David, A., Lebendiker, M., Benyamini, H., Izraeli, S., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STIL+protein+contains+intrinsically+disordered+regions+that+mediate+its+protein-protein+interactions.">The STIL protein contains intrinsically disordered regions that mediate its protein-protein interactions.</a> Chemical Communications. 50, 5245-5247 (2013).</li><li>Izraeli, S., Lowe, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SIL+gene+is+required+for+mouse+embryonic+axial+development+and+left-right+specification.">The SIL gene is required for mouse embryonic axial development and left-right specification.</a> Nature. 399 (6737), 691-694 (1999).</li><li>Izraeli, S., Colaizzo-Anas, T., Bertness, V. L., Mani, K., Aplan, P. D., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+SIL+gene+is+correlated+with+growth+induction+and+cellular+proliferation.">Expression of the SIL gene is correlated with growth induction and cellular proliferation.</a> Cell Growth Differ. 8 (11), 1171-1179 (1997).</li><li>Arquint, C., Sonnen, K. F., Stierhof, Y. -. D., Nigg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Cell-cycle-regulated+expression+of+STIL+controls+centriole+number+in+human+cells.">a Cell-cycle-regulated expression of STIL controls centriole number in human cells.</a> Journal of Cell Science. 125 (5), 1342-1352 (2012).</li><li>Tang, C. J., Lin, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+microcephaly+protein+STIL+interacts+with+CPAP+and+is+required+for+procentriole+formation.">The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.</a> Embo J. 30 (23), 4790-4804 (2011).</li><li>Vulprecht, J., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIL+is+required+for+centriole+duplication+in+human+cells.">STIL is required for centriole duplication in human cells.</a> Journal of Cell Science. 125 (5), 1353-1362 (2012).</li><li>Campaner, S., Kaldis, P., Izraeli, S., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sil+phosphorylation+in+a+Pin1+binding+domain+affects+the+duration+of+the+spindle+checkpoint.">Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.</a> Mol Cell Biol. 25 (15), 6660-6672 (2005).</li><li>Kasai, K., Inaguma, S., Yoneyama, A., Yoshikawa, K., Ikeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCL/TAL1+interrupting+locus+derepresses+GLI1+from+the+negative+control+of+suppressor-of-fused+in+pancreatic+cancer+cell.">SCL/TAL1 interrupting locus derepresses GLI1 from the negative control of suppressor-of-fused in pancreatic cancer cell.</a> Cancer Res. 68 (19), 7723-7729 (2008).</li><li>Chaturvedi, P., Sudakin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chfr+regulates+a+mitotic+stress+pathway+through+its+RING-finger+domain+with+ubiquitin+ligase+activity.">Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.</a> Cancer Res. 62 (6), 1797-1801 (2002).</li><li>Olaussen, K. a., Commo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+proapoptotic+effects+of+the+two+tyrosine+kinase+inhibitors+pazopanib+and+lapatinib+on+multiple+carcinoma+cell+lines.">Synergistic proapoptotic effects of the two tyrosine kinase inhibitors pazopanib and lapatinib on multiple carcinoma cell lines.</a> Oncogene. 28 (48), 4249-4260 (2009).</li><li>Reingewertz, T. H., Britan-Rosich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Vif&#8211;A3G+interaction+using+peptide+arrays:+A+basis+for+anti-HIV+lead+peptides.">Mapping the Vif&#8211;A3G interaction using peptide arrays: A basis for anti-HIV lead peptides.</a> Bioorganic &amp; Medicinal Chemistry. 21 (12), 3523-3532 (2013).</li><li>Katz, C., Benyamini, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+interaction+between+the+antiapoptotic+Bcl-2+family+proteins+and+the+proapoptotic+protein+ASPP2.">Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.</a> Proc Natl Acad Sci U S A. 105 (34), 12277-12282 (2008).</li><li>Katz, C., Zaltsman-Amir, Y., Mostizky, Y., Kollet, N., Gross, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Basis+of+the+Interaction+between+Proapoptotic+Truncated+BID+(tBID)+Protein+and+Mitochondrial+Carrier+Homologue+2+(MTCH2)+Protein:+KEY+PLAYERS+IN+MITOCHONDRIAL+DEATH+PATHWAY.">Molecular Basis of the Interaction between Proapoptotic Truncated BID (tBID) Protein and Mitochondrial Carrier Homologue 2 (MTCH2) Protein: KEY PLAYERS IN MITOCHONDRIAL DEATH PATHWAY.</a> Journal of Biological Chemistry. 287 (18), 15016-15023 (2012).</li><li>Rosenberg, M. 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The authors have nothing to disclose.

Peptidarray-Screening ist ein ausgezeichnetes Werkzeug für die Identifizierung der Bindungsstellen eines Zielproteins in seine Partner. Dieser Test ist schnell und einfach durchzuführen und die Ergebnisse können in einem oder zwei Tagen erhalten werden. Peptidarray Screening ist vielseitig und kann für viele Zwecke verwendet werden. Es kann für die Charakterisierung von Übersetzungs- Modifikationen wie Phosphorylierung und Identifizieren Substrate von Kinasen eingesetzt werden. Zum Beispiel: der FLT3-Kinase, die v...

Protein-Protein-Wechselwirkungen vermitteln die meisten Prozesse in der lebenden Zelle. Impaired Protein-Interaktionen können in Krankheit führen, was Protein-Interaktionen wichtige Angriffspunkte für Arzneimittel. Es ist daher sehr wichtig, diese Interaktionen auf molekularer Ebene zu verstehen. Protein-Wechselwirkungen sind mit einer Vielzahl von Techniken, von der zellulären und biochemischen Tests, quantitative biophysikalische Assays untersucht, und diese können mit vollständigen Proteine ​​durchgeführt werden, entweder mit Proteindomänen oder mit Peptiden. Peptide dienen als ausgezeichnete Werkzeuge, um Protein-Wechselwirkungen zu untersuchen. Dies ist, weil Peptide können leicht synthetisiert werden und ermöglichen die Fokussierung auf einen bestimmten Interaktionsstelle einerseits und auf mehreren Protein-Targets in einem Hochdurch Weise auf der anderen Seite 1,2. Peptidarray Screening ist eine schnelle, einfache Verfahren zur Gewinnung einer großen Menge von Daten über die Wechselwirkungen eines Zielproteins mit zahlreichen Partnern in einer kurzen Zeit durchzuführen 3. Im Gegensatz zu anderen biochemischen oder biophysikalischen Methoden zur Detektion und Analyse von Protein-Protein-Wechselwirkungen, Peptid-Array-Screening erfordert eine sehr geringe Konzentration an Protein und sehr schwache Bindung zu detektieren. Peptid-Arrays können für viele Anwendungen in der Peptid-Protein-Wechselwirkungen, wie Mapping von Protein-Protein oder Rezeptor-Ligand-Interaktionsstellen 4, Homo- oder Hetero-Oligomerisierung Schnittstellen verwendet werden, kennzeichn Antikörper Epitope 5, studiert Enzymaktivitäten 6 und hohen Durchsatz struktur Aktivitäts-Beziehung (SAR) studiert 7. Für eine eingehende Bewertung Peptidarray Screening siehe Katz et al. 4 Verschiedene Arten von Peptidarrays existieren derzeit. Es gibt zwei Hauptstrategien für die Herstellung von synthetischen Peptidarrays: Synthese der Peptide vor der Befestigung an dem festen Träger oder der Synthese von Peptiden direkt an den festen Träger, vor allem mit Hilfe der SPOT-Technik 4,8. DiePeptide auf dem festen Träger in der Regel von 9-Fluorenylmethyloxycarbonyl (Fmoc) Chemie 8 synthetisiert. Unter den gemeinsamen Syntheseschemata sind Peptid Befestigung durch den N-Terminus (zB JPT Pepstar Arrays 9) und Peptid-Anlage durch den C-Terminus (zB PEPSCAN pepchip Arrays 10, JPT pepspot Arrays 9 und INTAVIS celluspot Arrays 11) 4. Der feste Träger kann variieren und damit auch die Chemie der Peptidkupplung zu. Cys-terminierten Peptide können über die Thiolgruppe 12 auf Glasobjektträgern befestigt werden. Der N-Terminus eines Peptids kovalent an eine Hydroxylgruppe an der Cellulose-Membran, durch Veresterung der Aminosäure gebunden befestigt 8. Hier stellen wir ein detailliertes Protokoll für das Screening von Peptid-Arrays als ein Verfahren zur Untersuchung von Protein-Protein-Wechselwirkungen. Das Array verwendeten wir die Celluspots Array, das ein Mikroarray mit großen amo istunt von Flecken (Duplikate bis 384 Spots) auf einer kleinen Cellulosemembran durch einen Glasträger unterstützt. Dies ermöglicht das Arbeiten mit geringen Mengen an Protein und Antikörper und erhalten erhebliche Menge an Daten pro einzigen Experiment. Dieses Array enthält auch eine hohe Dichte, die Peptid-Detektion mit geringer Affinität bindenden ermöglicht. Das Array wurde zur Kartierung des STIL-CHFR Interaktion, die sehr wichtig für die normale Zellproliferation 13 Steuerung verwendet wird. Unkontrollierte Wechselwirkung zwischen den beiden Proteinen zur Entwicklung von Krebs. Durch die Abbildung dieser Wechselwirkung fanden wir die spezifische Bindungsstelle und Bindungsreste 14. Dies ebnet den Weg für die Entwicklung rationeller Inhibitoren, die diese Protein-Protein-Wechselwirkung hemmen.

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:231px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptide array</td>
			<td>INTAVIS Bioanalytical Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">His-probe Antibody (H-3) HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8036 HRP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-ECL Kit</td>
			<td>Biological industries</td>
			<td>20-500-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skim Milk</td>
			<td>Becton, Dickinson and Company</td>
			<td style="width:119px;">232100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LAS-3000 camera&#160;</td>
			<td>FUJI film</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction sites using peptide arrays

Protein-Protein-Wechselwirkungen vermitteln die meisten Prozesse in lebenden Zellen und die Kontrolle der Homöostase des Organismus. Impaired Protein-Interaktionen können in Krankheit führen, was Protein-Interaktionen wichtige Angriffspunkte für Arzneimittel. Es ist daher sehr wichtig, diese Interaktionen auf molekularer Ebene zu verstehen. Protein-Wechselwirkungen sind mit einer Vielzahl von Techniken, von der zellulären und biochemischen Tests, quantitative biophysikalische Assays untersucht, und diese können mit vollständigen Proteine ​​durchgeführt werden, entweder mit Proteindomänen oder mit Peptiden. Peptide dienen als ausgezeichnete Werkzeuge, um Protein-Wechselwirkungen zu studieren, da Peptide können leicht synthetisiert werden und ermöglichen die Konzentration auf bestimmte Interaktionsstellen. Peptidarrays ermöglichen die Identifizierung der Interaktionsstellen zwischen zwei Proteinen sowie Screening nach Peptiden, die das Zielprotein für therapeutische Zwecke binden. Sie erlauben auch einen hohen Durchsatz SAR-Studien. Zur Identifizierung der Bindungsstellen, eine typical Peptidanordnung enthält in der Regel teilweise überlappenden Peptiden 10-20 Resten der vollen Sequenzen von einem oder mehreren Partnerproteine ​​der gewünschten Zielprotein. Screenen des Arrays zur Bindung des Zielproteins zeigt die Bindungspeptide, die den Bindungsstellen in den Partnerproteine, in einer einfachen und schnellen Verfahren mit nur geringen Menge an Protein. In diesem Artikel beschreiben wir ein Protokoll für das Screening von Peptidarrays für die Abbildung der Interaktionsstellen zwischen einem Zielprotein und seine Partner. Die Peptidanordnung ist auf der Basis der Sequenzen der Partnerproteine ​​unter Berücksichtigung ihrer Sekundärstruktur ausgebildet. Die in diesem Protokoll verwendeten Arrays waren Celluspots Arrays INTAVIS Bioanalytische Instrumente vorbereitet. Das Array wird blockiert, um unspezifische Bindung zu verhindern und anschließend mit dem untersuchten Protein inkubiert. Nachweis unter Verwendung eines Antikörpers offenbart die Bindungspeptide, die den spezifischen Wechselwirkungsstellen zwischen den Proteinen.

STIL ist ein sehr wichtiger zentrosomalen Protein. Es steuert die normale Zellteilung und Zellproliferation 13,15 - 19. STIL interagiert mit mehreren Proteinen, 18,20,21, und die meisten Interaktionen durch seinen zentralen Teil, die ein intrinsisch ungeordneten Bereich (IDR) 14 auf. Wir haben eine Reihe von Peptiden aus dem STIL Bindungsprotein CHFR abgeleitet sind. CHFR ist ein Tumorsuppressor, der in Reaktion auf Stress induzierten mitotischen 22. Da die Struktur der Bindun...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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

Ermittlung von Protein-Protein-Interaktionsstellen unter Verwendung von Peptid-Arrays

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

identifying-protein-protein-interaction-sites-using-peptide-arrays

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17.9K Aufrufe.  The Hebrew University of Jerusalem. Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Serie: Identifying Protein-protein Interaction Sites Using Peptide Arrays

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitin Basierend Membrane Yeast Two-Hybrid (Mythos) System: Ein wirksames Instrument zur Ermittlung von Protein-Protein-Wechselwirkungen

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Forschung

Biologie

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)

Bimolekularen Fluoreszenz Komplementation (BIFC) Assay für Protein-Protein-Wechselwirkungen in Onion Zellen unter Verwendung des 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 ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging Protein-Protein-Wechselwirkungen In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenese und funktionale Analyse von Ionenkanälen heterolog in Säugerzellen exprimierten

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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 ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Durchflusszytometrieanalyse bimolekularer Fluorescence Komplementierung: A High Throughput Quantitative Methode zur Protein-Protein Interaction Study

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Identifizierung von Protein-Protein-Wechselwirkung in<em&gt; Drosophila</em&gt; Adult Heads von Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Spezifitätsanalyse von Protein Lysin Methyltransferasen Mit SPOT Peptidarrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genomweite Protein-Protein-Wechselwirkungen durch Screening Protein-Fragment Komplementierungsassay (PKA) in lebenden Zellen

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...