This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1. Probe Vorbereitung <ol><li> Kultur und Reinigung der V5-Fusionskinase-Sonden verwendet werden, um Wechselwirkungen mit anderen Proteinen zu untersuchen, wie folgt: <ol><li> Verwenden Sie frisch ausgestrichenen Hefestamm Y258 (MATa pep4-3, his4-580, ura3-53, leu2-3,112), die V5-Fusionsprotein (von GATEWAY Vektor pYES-DEST52 ausgedrückt). Verwenden Sie das isolierte Protein als Sonde für die Mikroarrays. Platte die Hefe auf synthetischem Komplett-Uracil (SC-Ura) / 2% Dextrose / Agar und wachsen bei 30 ° C für 3 Tage aus gefrorenen Kultur (-80 ° C Glycerolstammlösung). </li><li> Impfen Starterkulturen (5-20 ml) aus einer Einzelkolonie und über Nacht wachsen in Sc-Ura / 2% Dextrose auf Schüttelplattform (220-250 rpm) oder Rad bei 30 ° C. </li><li> Am folgenden Morgen inokulieren 400 ml des Sc-Ura / 2% Raffinose Kultur mit ausreichend Starterkultur auf eine endgültige OD 600 von 0,1. </li><li> Wachsen die Impfkulturen bis zu einer OD 600 von 0,6, gefolgt von Galactose-Induktion des V5-Kinase-fusion Konstrukt-Expression durch Zugabe einer Lösung von 3x Hefeextrakt / Pepton (YEP) mit 6% Galactose ergänzt durch Zugabe von genug, um das Induktionsmittel durch einen Faktor von 3 zu verdünnen, so dass die endgültige Konzentration der Galactose beträgt 2%. </li><li> Induzieren Zellen bei 30 ° C für 6 Stunden auf eine schwankende Plattform. Verwenden Sie einen 2 l-Erlenmeyerkolben, angemessene Belüftung sicherzustellen. </li><li> Ernten der Zellen unter Verwendung eines JA-10 (oder vergleichbaren) Rotors durch Drehen mit 400 ml Zellsuspension bei 1.000 g für 5 min bei 4 ° C. </li><li> Mit 50 ml eiskaltem PBS-Puffer und in einen 50-ml konischen Röhrchen Waschen der Zellen einmal. Dann wird noch einmal das Pellet in eiskaltem PBS-Puffer (ohne Reinigungsmittel oder andere Additive) für die Lyse und Transfer zum 2 verwendet ml Röhrchen für die Lyse-Kappe einrasten. </li><li> Drehen Sie die Zellen bei 20.000 · g für 1 min bei 4 ° C zu einem Pellet und Pipette entfernt Puffer. </li><li> Zeigen Röhrchen auf Eis und füllen Lyseschritt. </li><li> Lyse der Zellen (250-350 ul Pellet) mit 00,5 mm Zirkoniumoxidkügelchen in einer 1: 1: 1 Volumen des Zellpellets, Perlen und phosphatgepufferter Kochsalzlösung (PBS) Lysepuffer und Wirbel das Gemisch unter Verwendung eines Bewegungsplattform 3 mal in 2 min Intervallen bei 4 ° C. </li><li> Zentrifugieren Sie das Lysat bei 20.000 × g in einer Tischmikrozentrifuge für 10 min bei 4 ° C. </li><li> Pipette Stand in Polycarbonat Hochgeschwindigkeitszentrifugenröhrchen und Klärung durch ulracentrufugation für 30 Minuten bei 150.000 × g bei 4 ° C. </li><li> Übertragen die geklärte Lysat in ein Röhrchen vorgewaschen Ni 2+ Affinitätsharz (~ 100 &amp; mgr; l) und Inkubieren auf einem Nutator für 2 h bei 4 ° C zu erfassen Histidin (His) 6X markierten V5-Fusionsprotein ist. </li><li> Dreimal Waschen des Harzes für 10 Minuten bei 4 ° C mit Waschpuffer. Pelletierung des Harzes unter Verwendung einer Tisch-Zentrifuge 5 min bei 1000 × g bei 4 ° C und saugt den Überstand. In frischem Waschpuffer für jeden Wasch und senden Sie das Rohr Nutatorwinkel für Agitation während der Wäsche. </li><li> Nach dem Wasch steps abgeschlossen sind, wenden die gewaschene Harz in ein frisches G-25-Säule für die Elution. </li><li> Gelten 25 ul Elutionspuffer in einer schrittartigen Weise, beginnend mit 100 mM Imidazol und 500 mM Imidazol in 50 mM Inkremente für insgesamt 9 Fraktionen. </li><li> Assay Die gesammelten Fraktionen mittels Natriumdodecylsulfat-Polyacrylamidgelelektrophorese (SDS-PAGE) und Coomassie-Färbung, um die am stärksten angereicherten Fraktion (en) zur Verwendung in dem Assay zu identifizieren und fügen Glycerin zu 30% und bei -80 ° C bis in den verwendeten Assay. </li></ol></li></ol> 2. Beobachtung der Arrays HINWEIS: Bitte beachten Sie Schritt 2.6 dieses Abschnitts, um die Antikörperlösung vor dem Test vorzubereiten. <ol><li> Um Wechselwirkungen zu erfassen, verdünnen das V5-Fluorophor konjugierten Antikörper (dh AlexaFluor 647) bis 260 ng / ml in Sondenpuffer und mischen durch Schütteln. HINWEIS: Bereiten Sie dieses Antikörperlösung 30 Minuten vor der Anwendung und legen Sie den Schlauch auf einem nutatoder oder ein Rad vollständige Vermischung und eine homogene Suspension von Antikörper zu gewährleisten. </li><li> Verdünne die V5-Fusionsprotein Sonde über einen Konzentrationsbereich von 5-500 &amp; mgr; g / ml. HINWEIS: Optimiert für jedes Protein-Protein-Interaktionsassays, mit Sondenpuffer. Optimierung beinhaltet das Hinzufügen von mehr Sonden an Puffer zu sondieren. Typischerweise 10 &amp; mgr; g / ml wird als Ausgangspunkt verwendet und angepasst basierend auf der Signalstärke. </li><li> Entfernen der Proteinarrays aus dem Tiefkühler (-20 ° C) und bringen auf 4 ° C im Kühlraum unmittelbar vor der Verwendung. </li><li> In Blocking-Puffer direkt auf die Objektträgerhalter, die die Protein micorarrays und decken den Spitzen mit Parafilm, um ein Auslaufen zu verhindern. Blockieren die Arrays in Blockierungspuffer für 1 Stunde unter Schütteln bei 50 UpM auf einer Stufe bei 4 ° C. </li><li> Nach dem Blockieren übertragen die Arrays einer befeuchteten Kammer auf 4 ° C gekühlt, und geben 90 ul der verdünnten Probe direkt auf der Array-Oberfläche. Überlagern die arrays mit einem erhöhten Schlupf Heber und Inkubation statisch (ohne Schütteln) in der feuchten Kammer bei 4 ° C für 1,5 Std. </li><li> 3 mal in drei 50 ml konische Röhrchen waschen die Arrays für jeweils 1 min in Puffer Sonde. Fügen Sie die Folie, um konische Röhrchen, die genug vorgekühlte Sondenpuffer, um die Folie vollständig umhüllen. Lassen Sie den Lifter Schlupf sanft abrutschen des Proteinmikroarray (keine Gewalt aus, da dies zu Schäden an der Array-Oberfläche zur Folge haben). </li><li> Übernehmen Sie die Antikörper-Lösung direkt auf Array sofort nach Abschluss des Wasch (Schritt 2.5) und Überlagerung mit einem erhöhten Heber Schlupf wie zuvor. Die Arrays 30 min Inkubation bei 4 ° C in der feuchten Kammer. </li><li> Führen die gleiche Waschschritt wie zuvor (3 mal 1 min in Puffer Sonde) und Spin in einem 50 ml konischen Röhrchen bei 800 x g in einer Tischzentrifuge 5 min bei Raumtemperatur. Der Luft trocknen die Arrays in einem Objektträgerhalter in der Dunkelheit für 30 Minuten vor der Anordnung bei 647 nm gescannt. </li></ol>

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The authors declare that they have no competing financial interests.

Das Protokoll vorgestellt wurde ursprünglich unter Verwendung von 85 einzigartigen Hefe-Proteinkinase-V5-Fusionsproteinen die Bindungsaktivität gegenüber verschiedenen und verwandte Familien von Hefe-Proteinkinasen, was zur Identifizierung von neuen Kinase-Interaktionsnetz in vivo 1 zu vergleichen. Als aufstrebender Proteom-Profiling-Technologie, die Entwicklung von High Throughput (HTP) Screening der Proteome mit Protein-Microarrays auf Peptidbibliotheken und eukaryotischen und prokaryotischen Mo...

Die Notwendigkeit, die globale Analysen der Proteinbiochemie und Bindungsaktivität in vivo durchzuführen in der Entwicklung neuer Verfahren zum Profilieren von Protein-Protein-Wechselwirkungen (PPI) und die post-translationalen Modifikationen von ganzen Proteomen 1,3-8 geführt. Protein-Mikroarrays werden als funktionelle Protein-Microarrays mit voller Länge funktionelle Proteine ​​4-6,8,9 oder analytische Protein-Microarrays 10,11 Antikörper enthält, hergestellt. Sie sind so konstruiert, um eine hohe Dichte von Proteinen auf Mikroskop-Objektträgern mit einer Vielzahl von Oberflächenchemie angeordnet enthalten, um eine Vielzahl von zum Leiten von weitreichenden biochemischen Analysen erforderlichen 12 Versuchsbedingungen zu erleichtern. Nitrocellulose und Aldehyd Oberflächenchemie für die chemische Anbindung durch Lysin oder Affinität Befestigungsmethoden, wie beispielsweise Nickel-Chelat-Objektträger zur Befestigung His-markierte Proteine ​​und Glutathion zur Affinitäts Befestigung unter anderem 13. </p&gt; Die Verwendung von funktionellen Protein-Mikroarrays, um Protein-Protein-Interaktionen zu detektieren erfordert den Zugriff auf einen hochwertigen funktionellen Proteinbibliothek 14. S. cerevisiae ist zugänglich Herstellung einer solchen Bibliothek durch die Paarung von hoher Kopienaffinitätsmarkierten Proteinkonstrukte mit Hochdurchsatz-chromatographischen Reinigung Techniken. Die überwiegende Mehrheit des Hefegenoms sequenziert wurde und fast die gesamte Proteoms kann aus einem high-copy-Plasmid zur Reinigung exprimiert und biochemischen Analysen 12. Sobald die Proteine ​​gewonnen und in 384-Well-Format angeordnet sind, werden sie auf einen Objektträger so dass für eine schnelle parallele multiparametrischer biochemische Analytik und Bioinformatik Abfrage 8,14-16 gedruckt. Protein-Mikroarrays für enzymatische Assays und Wechselwirkungen mit Proteinen, Lipiden, kleine Moleküle und Nukleinsäuren, unter vielen anderen Anwendungen eingesetzt. Die Zugänglichkeit von Proteinen auf der Oberfläche des ProteomsAnordnungen machen, sich auf andere Arten von analytischen Nachweis einschließlich Immunaffinitäts, Oberflächenplasmonresonanz, Fluoreszenz und viele andere Techniken. Außerdem ermöglicht sie die Feinsteuerung der experimentellen Bedingungen ab, wo es schwer, in vivo tun. Ziel dieses Protokolls ist es, die angemessene Verwendung der funktionellen Protein-Microarrays, um Protein-Protein Wechselwirkungen erkennen zu demonstrieren. Diese Anwendung ermöglicht die Hochdurchsatz parallel biochemische Analyse von Protein-Bindungsaktivitäten unter Verwendung eines hoch gereinigten Analyten (Protein) von Interesse. Ein C-terminal (carboxyterminalen) Markierte V5-Fusions-Köderprotein von Interesse aus einem high-copy-Plasmid in einem Hefestamm zur Proteinreinigung optimiert produziert. C-terminalen Markierungs sichergestellt, dass das Volllängen-Protein übersetzt worden ist. Das in dieser Studie verwendete Protein ist TDA1-V5-Fusionsprotein-Kinase, die mit gereinigtem Nickel-Affinitätsharz über eine His6X tag. Die TDA1-V5-Fusions Baut wird durch serielle Elution mit einem Imidazol-Gradienten, um die am stärksten angereicherten Fraktion für den Einsatz im Assay Eluieren gereinigt.

<table border="0" cellpadding="0" cellspacing="0" width="855">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Petri Dishes</td>
			<td style="width:180px;">VWR</td>
			<td>NC-10747</td>
			<td>or comparable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto yeast extract</td>
			<td style="width:180px;">Difco</td>
			<td>0217-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto peptone</td>
			<td style="width:180px;">Difco</td>
			<td>0118-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:180px;">Difco</td>
			<td>0140-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dextrose</td>
			<td style="width:180px;">Sigma</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Raffinose</td>
			<td style="width:180px;">Sigma</td>
			<td>R0514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Galactose</td>
			<td style="width:180px;">Sigma</td>
			<td>G0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sc-Ura drop out media</td>
			<td style="width:180px;">MP Bio</td>
			<td>114410622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Small Culture tubes</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Large Erlenmeyer Flask</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge JA-10 rotor</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge tubes (500 mL)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Falcon tube (50 mL)</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PBS Tablets</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Tubes(2ml screw cap tube)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Machine</td>
			<td style="width:180px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">116004500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Zircon Beads</td>
			<td style="width:180px;">BioSpec</td>
			<td align="right" style="width:119px;">11079105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Triton X100</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DTT</td>
			<td style="width:180px;">Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">MgCl2</td>
			<td style="width:180px;">Sigma</td>
			<td>M8266&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">NaCl</td>
			<td style="width:180px;">Sigma</td>
			<td>S3014&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Imidazole (pH = 7.4)</td>
			<td style="width:180px;">Sigma</td>
			<td>I5513&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PMSF</td>
			<td style="width:180px;">Sigma</td>
			<td>93482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Complete Inhibitor Cocktail (EDTA Free)</td>
			<td style="width:180px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Phosphatase inhibitor cocktail 1</td>
			<td style="width:180px;">sigma</td>
			<td>P2850</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSA</td>
			<td style="width:180px;">Sigma</td>
			<td>A2153&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Tween-20</td>
			<td style="width:180px;">Sigma</td>
			<td>P1379&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ATP</td>
			<td style="width:180px;">Sigma</td>
			<td>A1852&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Shaking Incubator (30 &#176;C)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nickel Affinity Resin</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R901-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">G25 column</td>
			<td style="width:180px;">GE life sciences</td>
			<td>27-5325-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nutator</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">SDS-PAGE Gel (NuPage)</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Coomassie Blue Stain</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R960-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa647 Tagged monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>451098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Protein Microarrays Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td>PAH0525013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Genepix Scanner</td>
			<td style="width:180px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:267px;">Lifter Slips</td>
			<td style="width:180px;">Thomas Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:291px;">http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td>
		</tr>
		<tr height="124">
			<td height="124" style="height:124px;width:267px;">Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT, 
			2 mM MgCl2, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&#160; (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS to obtain the desired volume of blocking buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Probe Buffer: 2 mM MgCl2, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl,500 &#956;M ATP (for kinases), 1% BSA</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:267px;">Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl2</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:267px;">Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl2, 500 mM NaCl, 50 &#8211; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Before you begin, it is important to note the concentration gradient of imidazole (50 &#8211; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:267px;">3x YEP +6% galactose: Dissolve 30 g of yeast extract and&#160; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;galactose should not be autoclaved</td>
		</tr>
	</tbody>
</table>

probing high-density functional protein microarrays to detect protein-protein interactions

Hochdichte funktionelle Protein-Microarrays ~ 4.200 rekombinanten Hefe Proteine ​​enthalten, werden für Kinase-Protein-Protein-Wechselwirkungen unter Verwendung eines affinitätsgereinigten Hefe Kinase-Fusionsproteins, enthaltend ein V5-Epitop-Tag zum Auslesen sucht. Gereinigte Kinase wird durch die Kultur eines Hefestammes für eine hohe Kopienproteinproduktion, der ein Plasmid enthält eine Kinase-V5-Fusionskonstrukt unter einem GAL induzierbarer Promotor optimiert erhalten. Die Hefe wird in restriktiven Medien mit einem neutralen Kohlenstoffquelle für 6 Stunden, gefolgt von der Induktion mit 2% Galactose gezüchtet. Anschließend wird die Kultur geerntet und Kinase unter Verwendung von Standard affinitätschromatographischen Methoden, eine hoch gereinigte Protein-Kinase für die Verwendung in Assays zu erhalten, gereinigt. Die gereinigte Kinase wird mit Kinase-Puffer auf einen geeigneten Bereich für den Test verdünnt, und die Protein-Mikroarrays werden vor der Hybridisierung mit dem Protein-Mikroarray blockiert. Nach der Hybridisierung werden die Arrays mit monoklonalen V5 ANTIB sondiertody um Proteine ​​durch die Kinase-V5-Protein gebunden zu identifizieren. Schließlich werden die Arrays mit einem Standard-Microarray-Scanner abgetastet und Daten für nachgelagerte Informatik Analyse 1,2 extrahiert, um ein hohes Vertrauen Reihe von Protein-Interaktionen für Downstream-Validierung in vivo zu bestimmen.

Das Protein-Protein-Wechselwirkung-Aktivität wurde mit einem Standard-Chip-Leser zu beurteilen, die TDA1-V5 Protein-Kinase-Fusionskonstrukt als Köderprotein gegen eine Hefe funktionelle Protein-Microarray, das etwa 4.200 einzigartige S. beobachtet cerevisiae GST-Fusionsproteinen. Weitere Abfrage mit der Genepix Software ergab eine Vielzahl von Bindungsereignissen unterschiedlicher Intensität. Die Affinität wurde aus dem abgestuften Intensität des von einem monoklonalen V5-Fluorophor-konjugierten A...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Verwendung von Protein-Mikroarrays enthält fast die gesamte S. cerevisiae Proteom ist für schnelle unvoreingenommene Abfragetausende von Protein-Protein-Wechselwirkungen in parallel sondiert. Dieses Verfahren kann für die Protein-Kleinmolekül, posttranslationale Modifikation und andere Assays mit hohem Durchsatz verwendet werden.

Probing hoher Dichte Funktionale Protein-Microarrays, um Protein-Protein Wechselwirkungen erkennen

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

probing-high-density-functional-protein-microarrays-to-detect-protein

Research

JoVE Journal

Biology

7.9K Aufrufe.  Stanford University.  Verwendung von Protein-Mikroarrays enthält fast die gesamte S. cerevisiae Proteom ist für schnelle unvoreingenommene Abfragetausende von Protein-Protein-Wechselwirkungen in parallel sondiert. Dieses Verfahren kann für die Protein-Kleinmolekül, posttranslationale Modifikation und andere Assays mit hohem Durchsatz verwendet werden. 

Serie: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

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

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

In-vivo-Detektion von Protein-Protein-Interaktionen auf Micro-gemusterten Oberflächen

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

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, ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Ein Protokoll für die Identifizierung von Protein-Protein-Wechselwirkungen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung, Immunpräzipitation, Quantitative Massenspektrometrie und Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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 ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

Mit Cokultur zur Erkennung chemisch vermittelte Interspezies-Interaktionen

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Protein-Protein-Wechselwirkungen durch Bimolekulare Fluoreszenz-Komplementation in Tabak-Protoplasten-und Blätter-Visualisierte

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

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 ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

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

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 ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Entwicklung von Inhibitoren von Protein-Protein-Wechselwirkungen durch REPLACE: Anwendung auf die Konzeption und Entwicklung Nicht ATP Competitive CDK-Inhibitoren

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...