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. Préparation de la sonde <ol><li> Culture et purifier les sondes kinase V5-fusion utilisées pour examiner les interactions avec d&#39;autres protéines comme suit: <ol><li> Utilisez fraîchement striée souche de levure Y258 (MATa pep4-3, his4-580, ura3-53, leu2-3,112) contenant des protéines V5-fusion (exprimé de Gateway vecteur pYES-DEST52). Utilisez la protéine isolée comme sonde sur les puces. Plaque sur la levure synthétique complet-uracile (Sc-Ura) / 2% de dextrose / agar et de croître à 30 ° C pendant 3 jours de culture congelée (-80 ° C glycérol stock). </li><li> Inoculer des cultures de démarrage (5-20 ml) à partir d&#39;une seule colonie et de croître pendant la nuit dans Sc-Ura / 2% de dextrose à secouer la plate-forme (220-250 rpm) ou de la roue à 30 ° C. </li><li> Le lendemain matin, inoculer 400 ml de la Sc-Ura / 2% de la culture de raffinose avec la culture de démarrage suffisant pour une DO finale 600 de 0,1. </li><li> Cultivez les inoculums à DO 600 de 0,6 suivie par induction par le galactose de la fu V5-kinasesion construire expression par addition d&#39;une solution d&#39;extrait de levure 3x / Peptone (YEP) additionné de 6% de galactose, en suffisant pour diluer le support d&#39;induction d&#39;un facteur 3 en ajoutant de sorte que la concentration finale de 2% de galactose est. </li><li> Induire les cellules à 30 ° C pendant 6 heures sur une table d&#39;agitation. Utilisez un L Erlenmeyer de 2 pour assurer une aération appropriée. </li><li> Récolte des cellules en utilisant un rotor JA-10 (ou comparables), la rotation 400 ml de suspension de cellules à 1000 g pendant 5 min à 4 ° C. </li><li> Laver les cellules une fois avec 50 ml de glace tampon PBS froid et transférer dans un tube conique de 50 ml. Laver le culot de nouveau dans un tampon PBS glacé (sans détergents ou d&#39;autres additifs) utilisés pour la lyse et le transfert à 2 ml à bouchon tubes de lyse. </li><li> Faites tourner les cellules à 20.000 g pendant 1 min à 4 ° C à une pastille et la pipette loin le tampon. </li><li> Placer les tubes sur la glace et passez à l&#39;étape de lyse. </li><li> Lyse des cellules (250-350 pi granulés) avec 00,5 mm billes de zircone dans un 1: 1: volume de culot cellulaire, des perles, et une solution saline tamponnée au phosphate (PBS) 1 tampon de lyse et le vortex le mélange en utilisant une plate-forme d&#39;agitation 3 fois à intervalles de 2 min à 4 ° C. </li><li> Centrifuger le lysat à 20.000 xg dans une centrifugeuse de table pendant 10 min à 4 ° C. </li><li> Pipette surnageant dans le tube de centrifugeuse en polycarbonate à haute vitesse et de clarifier par ulracentrufugation pendant 30 minutes à 150 000 g à 4 ° C. </li><li> Transférer le lysat clarifié dans un tube contenant de la résine Ni 2+ prélavé d&#39;affinité (~ 100 ul) et incuber sur un nutator pendant 2 h à 4 ° C pour capturer l&#39;histidine (His) 6X protéine étiquetée V5-fusion. </li><li> Laver la résine trois fois pendant 10 min à 4 ° C avec le tampon de lavage. Pellet la résine en utilisant une centrifugeuse de table pendant 5 min à 1000 g à 4 ° C et aspirer le surnageant. Ajouter tampon de lavage fraîche pour chaque lavage et retourner le tube du nutator pour l&#39;agitation pendant le lavage. </li><li> Après la ste de lavageps sont complets, appliquer la résine lavée à une colonne G-25 frais pour l&#39;élution. </li><li> Appliquer 25 pi de tampon d&#39;élution d&#39;une manière par étapes en commençant avec 100 mM d&#39;imidazole à 500 mM d&#39;imidazole 50 mM par incréments pour un total de neuf fractions. </li><li> Doser les fractions recueillies en utilisant du gel de dodécylsulfate de sodium et de polyacrylamide (SDS-PAGE) et coloration de Coomassie pour déterminer la fraction (s) le plus fortement enrichi pour être utilisé dans l&#39;essai et ajouter du glycérol à 30% et stocker à -80 ° C jusqu&#39;à utilisation dans le dosage. </li></ol></li></ol> 2. Sonder les tableaux NOTE: S&#39;il vous plaît voir l&#39;étape 2.6 de cette section pour préparer la solution d&#39;anticorps avant de commencer l&#39;essai. <ol><li> Pour détecter les interactions, diluer l&#39;anticorps conjugué V5-fluorophore (c. AlexaFluor 647) à 260 ng / ml dans le tampon de la sonde et mélanger soigneusement par agitation. NOTE: Préparer cette solution d&#39;anticorps 30 min avant de les utiliser et placer le tube sur un nutatou ou la roue pour assurer un mélange complet et une suspension homogène d&#39;anticorps. </li><li> Diluer la sonde de protéines V5-fusion sur une plage de concentration de 5-500 pg / ml. REMARQUE: optimisée pour chaque essai d&#39;interaction protéine-protéine, en utilisant un tampon de la sonde. L&#39;optimisation consiste à ajouter plusieurs sondes pour sonder tampon. Typiquement, 10 ug / ml est utilisée comme point de départ et ajusté en conséquence en fonction de la puissance du signal. </li><li> Retirer les puces à protéines du congélateur (-20 ° C) et porter à 4 ° C au réfrigérateur juste avant l&#39;utilisation. </li><li> Ajouter un tampon bloquant directement le support de diapositives contenant les micorarrays de protéines et de couvrir le dessus avec du parafilm pour éviter les fuites. Bloquer les tableaux dans un tampon de blocage pendant 1 heure en agitant à 50 rpm sur une scène à 4 ° C. </li><li> Après blocage, les ensembles de transférer une chambre humidifiée refroidi à 4 ° C et ajouter 90 ul de sonde diluée directement à la surface du tableau. Superposer l&#39;arrays avec un bordereau de lifter soulevé et incuber statique (sans agitation) dans la chambre humide à 4 ° C pendant 1,5 heures. </li><li> Laver les tableaux 3 fois pendant 1 min dans un tampon de chaque sonde dans trois 50 ml tubes coniques. Ajouter la diapositive à tubes coniques contenant un tampon de la sonde pré-réfrigérés assez pour envelopper complètement la diapositive. Laisser le bordereau de levier pour doucement glisser hors de la puce de protéine (ne pas forcer off car cela pourrait entraîner des dommages à la surface de la matrice). </li><li> Appliquer la solution d&#39;anticorps directement au tableau immédiatement après avoir terminé le lavage (étape 2.5) et la superposition avec un glissement de levage soulevé comme avant. Incuber les matrices pendant 30 minutes à 4 ° C en chambre humide. </li><li> Effectuez la même étape de lavage comme avant (3 fois 1 min dans un tampon de la sonde), et de spin dans un tube conique de 50 ml à 800 xg dans une centrifugeuse de table pendant 5 min à température ambiante. Air-sécher les tableaux dans un support de diapositives dans l&#39;obscurité pendant 30 min avant de balayer le tableau à 647 nm. </li></ol>

<ol>
	<li>Fasolo, J., 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=Diverse+protein+kinase+interactions+identified+by+protein+microarrays+reveal+novel+connections+between+cellular+processes.">Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes.</a> Genes. 25, 767-778 (2011).</li><li>Zhu, X., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProCAT:+a+data+analysis+approach+for+protein+microarrays.">ProCAT: a data analysis approach for protein microarrays.</a> Genome biology. 7, R110 (2006).</li><li>Breitkreutz, A., 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=A+global+protein+kinase+and+phosphatase+interaction+network+in+yeast.">A global protein kinase and phosphatase interaction network in yeast.</a> Science. 328, 1043-1046 (2010).</li><li>Gavin, A. 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=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415, 141-147 (2002).</li><li>Jeong, J. S., 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=Rapid+identification+of+monospecific+monoclonal+antibodies+using+a+human+proteome+microarray.">Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.</a> Mol Cell Proteomics. 11 (6), (2012).</li><li>Krogan, N. J., 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=Global+landscape+of+protein+complexes+in+the+yeast+Saccharomyces+cerevisiae.">Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.</a> Nature. 440, 637-643 (2006).</li><li>Popescu, S. 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=Differential+binding+of+calmodulin-related+proteins+to+their+targets+revealed+through+high-density+Arabidopsis+protein+microarrays.">Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 4730-4735 (2007).</li><li>Zhu, H., 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=Global+analysis+of+protein+activities+using+proteome+chips.">Global analysis of protein activities using proteome chips.</a> Science. 293, 2101-2105 (2001).</li><li>Ptacek, J., 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=Global+analysis+of+protein+phosphorylation+in+yeast.">Global analysis of protein phosphorylation in yeast.</a> Nature. 438, 679-684 (2005).</li><li>Haab, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+arrays+in+cancer+research.+Molecular+&amp;+cellular+proteomics.">Antibody arrays in cancer research. Molecular &amp; cellular proteomics.</a> Mol Cell Proteomics. 4 (4), 377-383 (2005).</li><li>Kopf, E., Zharhary, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+arrays--an+emerging+tool+in+cancer+proteomics.">Antibody arrays--an emerging tool in cancer proteomics.</a> The international journal of biochemistry &amp; cell biology. 39, 1305-1317 (2007).</li><li>Berrade, L., Garcia, A. E., Camarero, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarrays:+novel+developments+and+applications.">Protein microarrays: novel developments and applications.</a> Pharmaceutical research. 28, 1480-1499 (2011).</li><li>Balboni, I., Limb, C., Tenenbaum, J. D., Utz, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microarray+surfaces+and+arraying+parameters+for+autoantibody+profiling.">Evaluation of microarray surfaces and arraying parameters for autoantibody profiling.</a> Proteomics. 8, 3443-3449 (2008).</li><li>Gelperin, D. M., 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=Biochemical+and+genetic+analysis+of+the+yeast+proteome+with+a+movable+ORF+collection.">Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.</a> Genes &amp; development. 19, 2816-2826 (2005).</li><li>Fasolo, J., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarrays.">Protein microarrays.</a> Methods Mol. Biol. 548, 209-222 (2009).</li><li>Im, H., Snyder, M., Coligan, J. E., 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=Preparation+of+recombinant+protein+spotted+arrays+for+proteome-wide+identification+of+kinase+targets.">Preparation of recombinant protein spotted arrays for proteome-wide identification of kinase targets.</a> Current protocols in protein science. 27, Unit 27 24 (2013).</li><li>MacBeath, G., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+proteins+as+microarrays+for+high-throughput+function+determination.">Printing proteins as microarrays for high-throughput function determination.</a> Science. 289, 1760-1763 (2000).</li></ol>

The authors declare that they have no competing financial interests.

Le protocole présenté a été effectuée en utilisant 85 des protéines kinase-V5 de fusion protéine de levure uniques pour comparer l&#39;activité de liaison à travers familles distinctes et liées des protéines kinases de la levure conduisant à l&#39;identification de nouveaux réseaux d&#39;interactions in vivo une kinase. En tant que technologie de profilage protéomique émergents, le développement de High Throughput (HTP) de dépistage du protéome en utilisant des puces à protéines a...

La nécessité de réaliser des analyses globales de biochimie des protéines et de l&#39;activité in vivo de liaison a abouti à l&#39;élaboration de nouvelles méthodes de profilage interactions protéine-protéine (IPP) et les modifications post-traductionnelles de protéomes entiers 1,3-8. Puces à protéines sont fabriqués comme puces fonctionnelles de protéines en utilisant des protéines fonctionnelles pleine longueur 4-6,8,9, ou des puces à protéines analytiques contenant des anticorps 10,11. Ils sont conçus pour contenir une grande densité de protéines disposées sur des lames de microscope avec une variété de compositions chimiques de surface pour faciliter une variété de conditions expérimentales requises pour effectuer biochimique large 12 analyses. Nitrocellulose et de la surface de l&#39;aldéhyde chimies pour la fixation chimique à travers lysine ou fixation d&#39;affinité des méthodes telles que nickel diapositives chélatés pour fixer les protéines et les glutathion His-étiqueté pour la fixation d&#39;affinité entre autres 13. </p&gt; L&#39;utilisation de puces à protéines fonctionnelles pour détecter les interactions protéine-protéine nécessite l&#39;accès à une bibliothèque de protéine fonctionnelle de haute qualité 14. S. cerevisiae est susceptible de produire une telle bibliothèque à travers le jumelage de haute affinité copie étiqueté constructions de protéines à haut débit avec des techniques de purification chromatographique. La grande majorité du génome de la levure a été séquencé et la quasi-totalité du protéome peut être exprimée à partir d&#39;un plasmide grand nombre de copies pour la purification et l&#39;analyse biochimique 12. Une fois que les protéines sont obtenus et disposés en format de 384 puits, ils sont imprimés sur une lame de microscope permettant une analyse biochimique rapide parallèle multi-paramétrique et l&#39;interrogatoire bioinformatique 8,14-16. microréseaux de protéines ont été utilisées pour les dosages enzymatiques et des interactions avec des protéines, des lipides, des petites molécules et des acides nucléiques, parmi beaucoup d&#39;autres applications. L&#39;accessibilité des protéines sur la surface du protéometableaux rendent prêtent à différents types de détection analytique, y compris immunitaire affinité, résonance plasmonique de surface, la fluorescence et de nombreuses autres techniques. En outre, il permet un contrôle précis de la condition expérimentale où il pourrait être difficile de le faire in vivo. L&#39;objectif de ce protocole est de démontrer l&#39;usage approprié des puces à protéines fonctionnelles pour détecter les interactions protéine-protéine. Cette application permet à l&#39;analyse biochimique parallèle à haut débit des activités de liaison de protéines en utilisant un analyte hautement purifiée (protéine) d&#39;intérêt. A C-terminal (carboxy-terminale) étiqueté V5-fusion protéine appât d&#39;intérêt est produit à partir d&#39;un plasmide grand nombre de copies dans une souche de levure optimisé pour la purification des protéines. C-terminal marquage assure que la protéine pleine longueur a été traduit. La protéine utilisée dans cette étude est TDA1-V5 protéine de fusion kinase, qui est purifiée en utilisant une résine d&#39;affinité de nickel via un tag His6X. La fusion construc TDA1-V5t est purifié par élution série en utilisant un gradient d&#39;imidazole pour éluer la fraction la plus hautement enrichi destiné à être utilisé dans le dosage.

<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

Haute densité des puces à protéines fonctionnelles contenant 4,200 ~ protéines de levure recombinantes sont examinées pour les interactions protéine-protéine kinase en utilisant une protéine de fusion purifiée par affinité de la kinase de levure contenant une étiquette V5-épitope de lecture. Kinase purifiée est obtenue par la culture d&#39;une souche de levure optimisé pour la production de protéines de haute copie hébergeant un plasmide contenant une fusion kinase-V5 construire sous un promoteur inductible GAL. La levure est cultivée dans des milieux restrictive avec une source de carbone neutre pendant 6 heures, suivi par induction avec 2% de galactose. Ensuite, la culture est récoltée et la kinase est purifiée en utilisant des techniques standards de Chromatographie d&#39;affinité pour obtenir une protéine kinase hautement purifiée pour une utilisation dans le dosage. La kinase purifiée est diluée avec un tampon de kinase à une gamme appropriée pour le dosage et les puces à protéines sont bloqués avant l&#39;hybridation avec le microréseau de protéines. Après l&#39;hybridation, les tableaux sont sondés avec des anticorps monoclonaux V5 antiborps d&#39;identifier les protéines liées par la protéine kinase-V5. Enfin, les tableaux sont numérisés à l&#39;aide d&#39;un scanner de puces à ADN standard, et les données sont extraites pour analyse informatique aval 1,2 à déterminer un niveau de confiance élevé définir des interactions des protéines pour la validation aval in vivo.

L&#39;activité de l&#39;interaction protéine-protéine a été observée à l&#39;aide d&#39;un lecteur de puce standard pour évaluer la TDA1-V5 protéine kinase Construction d&#39;une fusion protéine d&#39;appât contre une puce de protéine fonctionnelle de levure contenant environ 4200 uniques S. cerevisiae des protéines de fusion GST. Nouvel interrogatoire avec le logiciel Genepix révélé une multitude d&#39;événements de différentes intensités de liaison. L&#39;affinité a été évaluée à par...

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

En utilisant des puces à protéines contenant presque toute la S. cerevisiae protéome est sondé pour interrogatoire impartiale rapide de milliers d&#39;interactions protéine-protéine en parallèle. Cette méthode peut être utilisée pour la molécule de protéine-petite, la modification post-traductionnelle, et d&#39;autres dosages in à haut débit.

Sondage haute densité fonctionnels des puces à protéines pour détecter les interactions protéine-protéine

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 vues.  Stanford University.  En utilisant des puces à protéines contenant presque toute la S. cerevisiae protéome est sondé pour interrogatoire impartiale rapide de milliers d'interactions protéine-protéine en parallèle. Cette méthode peut être utilisée pour la molécule de protéine-petite, la modification post-traductionnelle, et d'autres dosages in à haut débit. 

Vidéo: 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-ubiquitine membrane à base de levure double hybride (MYTH) Système: Un outil puissant pour identifier les interactions entre protéines

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

Recherche

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 de détection des interactions protéine-protéine sur la micro-surfaces texturées

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.

Imagerie interactions protéine-protéine 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.

Un protocole pour l&#39;identification des interactions protéine-protéine Basé sur<sup&gt; 15</sup&gt; N marquage métabolique, immunoprécipitation, la spectrométrie de masse quantitative et d&#39;affinité de 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.

L&#39;analyse par cytométrie de flux par fluorescence bimoléculaire complémentation: une méthode quantitative à haut débit pour étudier l&#39;interaction protéine-protéine

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.

Utilisation de co-culture pour détecter les interactions interspécifiques par voie chimique

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.

Interactions protéine-protéine visualisés par fluorescence bimoléculaire complémentation en protoplastes de tabac et des feuilles

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.

Étudier les interactions protéine-protéine dans les cellules vivantes en utilisant le transfert d&#39;énergie de résonance de bioluminescence

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.

Identifier les sites d&#39;interaction protéine-protéine à l&#39;aide de peptides tableaux

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.

Développement d&#39;inhibiteurs de interactions protéine-protéine REMPLACER travers: Application à la conception et les inhibiteurs de CDK concurrentiel développement non-ATP

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