Ce travail a été soutenu par des subventions du CRSNG 386634. Nous tenons à remercier les membres du Screaton Lab pour des discussions utiles.

1. Préparation des réactifs, des assiettes et des cellules <ol><li> Ajouter 500 ml de tampon de lyse: Tris 25 mM pH 7,5, NaCl 150 mM, NaF 50 mM, EDTA 0,5 mM pH 8,0, 0,5% de Triton X-100, 5 mM de bêta-glycérophosphate, 5% de glycerol. Conserver à 4 ° C. Immédiatement avant l&#39;utilisation, ajouter 1 mM de dithiothréitol (DTT), 1 mM de fluorure de phénylméthyl sulfonyle (PMSF), vanadate de sodium et 1 mM. Après cette étape, PMSF est pas nécessaire dans tout tampon de rinçage. </li><li> Ajouter 20 ml de tampon kinase 10x: Tris 200 mM pH 7,5, 50 mM de bêta-glycérophosphate (FW 216), vanadate de sodium 2 mM. Aliquote de 1 ml tubes et conserver à -20 ° C. Immédiatement avant l&#39;utilisation, ajouter 5 mM de DTT. </li><li> Faire 20 ml de tampon 10x M-ATP: 300 um adénosine triphosphate (ATP), 66 mM de MgCl2, 33 mM MnCl 2. Aliquoter dans 1 ml tubes et conserver à -20 ° C. </li><li> Ajouter 100 ml de 2x du dodécylsulfate de sodium (SDS) de tampon de lyse: 1,5 g de base Tris, 20 ml de glycerol, 30 ml de H 2 O. Dissoudre et ajuster le pH à 6,8 avec HCl. Ajouter 40ml SDS à 10%, ajuster le volume à 100 ml. Ajouter 25 mg de bleu de bromophénol. </li><li> SPOT 4 pi de 25 ng / ul mammifère plasmide d&#39;expression codant pour un TPS-kinase 14 par puits dans des ensembles de plaques à 96 puits et les plaques d&#39;étiquettes en conséquence. Plaques d&#39;étanchéité et congeler à -20 ° C jusqu&#39;à utilisation. </li><li> 1-2 jours avant la transfection, les cellules HEK293T culture dans du milieu de Eagle modifié par Dulbecco complet (DMEM) + 10% de sérum bovin fœtal (FBS) et des antibiotiques dans un incubateur à 37 ° additionné de CO 2 (finale 5%). Passage à la trypsine et ne permettent pas le stock de devenir&gt; 80% de confluence lors de l&#39;expansion. Un minimum de 6 x 10 7 cellules sont nécessaires pour l&#39;écran (environ 3 x 15 cm boîtes de cellules HEK293T à 80% de confluence). </li></ol> 2. La transfection Remarque: Voir la Figure 1 pour un organigramme d&#39;ensemble du protocole. <ol><li> Si les plaques contenant des plasmides de kinases ont été gelés, les décongeler à la température ambiante, une centrifuger à 1900 g pendant 3 min pour recueillir l&#39;humidité au fond des puits. </li><li> Mélanger 8,6 ml de milieu de sérum réduit (par exemple, OPTI-MEM) avec 312,7 ul de réactif de transfection à base de lipides. Laisser reposer pendant 5 min. </li><li> Ajouter 10 ul de milieu de sérum réduit à chaque puits en utilisant un distributeur de liquide automatique équipé d&#39;une cassette de petit volume. </li><li> Ajouter 10 ul par puits de milieu de sérum / réactif de transfection mélange réduit de l&#39;étape 2.2 en utilisant un distributeur de liquide automatique équipé d&#39;une cassette de petit volume. Laisser reposer pendant 20 à 45 min. </li><li> Remettre en suspension les cellules 293T à 7,5 x 10 5 cellules / ml dans 80 ml ​​de DMEM complet. Ajouter 100 ul de suspension cellulaire (soit 7,5 x 10 4 cellules) par puits en utilisant un distributeur de liquide automatique équipé d&#39;une cassette de volume standard. </li><li> Vérifiez puits au microscope pour la distribution de cellules uniforme et retourner à l&#39;incubateur pendant 24 heures. </li></ol> 3. déroulant TPS-kinase <ol><li> Assurez-fresh: 4 mM de solution pervanadate en mélangeant 60 ul de 0,2 M vanadate de sodium avec 540 ul de H 2 O. Dans un second tube de mélange de 2,7 ul de peroxyde de 30% et 1,4 ml de PBS. Ajouter les deux solutions ensemble et laisser reposer pendant 15 min avant utilisation. </li><li> En utilisant une pipette multicanaux (ne pas utiliser un distributeur de liquide automatisé car elle crée trop de turbulence dans le puits) dispenser 2 pi de 0,25 M CaCl 2 dans chaque puits, suivi par 2,5 pi de la pervanadate préparé à l&#39;étape 3.1. Incuber chaque plaque à 37 ° C pendant 10 min, puis placer sur la glace. </li><li> Préparer 35 ml de tampon de lyse en ajoutant la TNT, PMSF, et vanadate de sodium comme indiqué à la section 1 (Préparation des réactifs). </li><li> Garder plaques sur de la glace, éliminer le milieu de chaque puits en utilisant un aspirateur à vide. Immédiatement ajouter 50 pl / puits de tampon de lyse froid de la glace en utilisant un distributeur de liquide automatisé muni d&#39;une cassette standard. Laisser reposer 30 min sur la glace de lyse (en option: peut arrêter là si nécessaire par plat d&#39;étanchéitée et le stockage à -80 ° C. Pour continuer, plaques dégel sur la glace). </li><li> Plaques de tourner à 1900 g pendant 3 min à 4 ° C. </li><li> Grattez les cellules de chaque puits à l&#39;aide d&#39;une pipette multicanaux et de transférer tous les contenus à dûment étiquetés plaques à 96 puits à fond en V. Plaques de tourner à 1900 g pendant 10 min à 4 ° C. </li><li> Lors de l&#39;essorage, remplissez glutathion revêtu plaques (une pour chaque plaque de 96 puits) avec 100 pl / puits de glace tampon de lyse froid (sans PMSF) comme un rinçage. Conserver les plaques sur de la glace. </li><li> Retirer des plaques à fond en V à partir de la centrifugeuse. Un à la fois, inverser les plaques de glutathion sur un évier pour secouer le tampon de lyse et tache sur une serviette en papier. Transfert de tampon de lyse à partir des plaques à fond en V aux plaques de glutathion en inclinant la plaque et à l&#39;aide d&#39;une pipette multicanaux, en faisant attention de ne pas perturber le culot au fond. Couvrir et laisser les plaques sur de la glace pendant au moins 2 heures à lier. </li><li> Près de la fin de l&#39;étape de liaison de deux heures, préparer une station de travail de la radioactivité, en veillant à til précautions de sécurité nécessaires sont en place pour le travail radioactifs. Set four d&#39;hybridation à 30 ° C. </li><li> Préparez 200 ml de tampon de lyse en ajoutant la TNT et de sodium vanadate comme indiqué à la section 1. PMSF est pas nécessaire à ce stade. </li><li> Inverser les plaques de glutathion sur un évier pour secouer le tampon de lyse et éponger sur un papier absorbant. Rincer puits 3x avec 100 tampon de lyse (sans PMSF). Ne pas laisser les puits sont assis à sec - garder dans rinçage jusqu&#39;à ce que prêt à procéder. </li><li> Préparer 55 ml de tampon kinase 1x (KB) en diluant le 10x stock et ajoutant la TNT comme indiqué à la section 1. Rincer les plaques une fois avec 50 pi de 1x KB aide d&#39;un distributeur de liquide automatisé muni d&#39;une cassette de volume standard. Laissez 1x KB dans les puits jusqu&#39;à ce que la solution A est prêt: <ol><li> Préparer la solution A en ajoutant 500 ug à 530 du substrat d&#39;intérêt, 500 ug de protéine basique de la myéline (MBP), 2,65 ml de 10x KB, 13,25 ul de DTT 1 M, et H 2 O 15,9 ml jusqu&#39;à. </li></ol></li&gt; <li> Un à la fois, inverser les plaques sur un évier pour enlever 1x KB rinçage, éponger sur du papier absorbant, et immédiatement ajouter 30 ul de la solution A l&#39;aide d&#39;un distributeur de liquide automatisé muni d&#39;une petite cassette de volume. Conserver les plaques sur de la glace. </li><li> Préparer la solution B dans la zone de travail de la radioactivité en ajoutant 2,5 ml 10x M-ATP, 32 P ATP de la 500 uCi, et H 2 O à 10 ml. <ol><li> Ajouter 20 pi de solution B par puits en utilisant une pipette à répétition qui aide à mélanger en raison de la force d&#39;éjection. Couvrir et incuber à 30 ° hybridation four C pendant 30 min. </li></ol></li><li> Après 30 minutes, transférer les plaques de retour à la glace. Ajouter 50 ul de tampon de lyse SDS 2x à chaque puits en utilisant une pipette multicanaux. Peut procéder à ce stade à l&#39;étape suivante, ou sceller les plaques avec une feuille d&#39;aluminium et conserver à -20 ° C jusqu&#39;à ce que pratique. </li></ol> 4. course, la coloration, et les gels de séchage Remarque: Tous les travaux doivent être effectués dans une désignation de zoneTed pour la radioactivité. <ol><li> Tournez sur l&#39;hybridation four et à 85 ° C. Plaques décongeler à température ambiante. Une fois que le four a atteint la température, plaques de transfert au four et incuber pendant 10 min pour dénaturer échantillons. </li><li> Charge: 26 puits gels pré-coulés à 15 ul de chaque mélange réactionnel à l&#39;aide d&#39;une pipette multicanaux pour remplir plusieurs puits à la fois. Il faut veiller à ce que tous les conseils alignent correspondant bien avant d&#39;ajouter échantillons. Exécutez gel à 150 V. Ne laissez pas le colorant de suivi (ligne bleue) fonctionne hors du fond du gel car il contient de l&#39;ATP non constituée en société. </li><li> Démonter les gels et couper l&#39;ATP non constituée en société (ligne bleue) à l&#39;aide d&#39;un scalpel ou le bord droit comme il se surexposer les films. Placez les gels dans des contenants étiquetés et couvrir avec Coomassie pendant 15 min. </li><li> Enlever la tache de Coomassie, brièvement rincer les gels avec de l&#39;eau, et d&#39;ajouter Destain solution. Destain les gels jusqu&#39;à ce que les protéines sont clairement visibles. Une bande de MBP et une bande pour le substrat devraitêtre visible pour chaque échantillon. </li><li> Pour sécher les gels: <ol><li> Couper une grande feuille de papier-filtre et le placer sur la sécheuse. </li><li> Mouiller une feuille de cellophane dans de l&#39;eau distillée jusqu&#39;à ce qu&#39;elle soit lisse et sans plis, et le placer sur le dessus du papier. </li><li> Disposez les gels sur le dessus de la feuille de cellophane, faire une note de l&#39;ordre de gels. Mouiller une deuxième feuille de cellophane et placer sur le dessus des gels. </li><li> Etaler toutes les bulles (un rouleau d&#39;étanchéité de la plaque fonctionne bien pour cela) pour une surface uniforme agréable. Fermer le clapet, tourner sur le vide, et sécher les gels pendant 3 heures à 80 ° C. </li></ol></li><li> Une fois que les gels sont secs, les exposer à un film XAR utilisant un écran d&#39;intensifier le signal. Enveloppez la cassette avec une pellicule ou un sac en plastique et sceller avec du ruban adhésif pour empêcher les gelées. Rangez la cassette à -80 ° C pendant la nuit. </li></ol> 5. Développer XAR Films <ol><li> Le lendemain, retirez la cassette du congélateur et laissez-les décongeler à la température ambiante. Développer le filmune chambre noire à l&#39;aide d&#39;un processeur de film selon les instructions du fabricant. Remarque: Examiner les films pour preuve de paires kinase-substrat. Une deuxième exposition plus longue peut également être utile pour détecter des événements de phosphorylation plus faibles. </li></ol>

<ol>
	<li>Meisenhelder, J., Hunter, T., van der Geer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphopeptide+mapping+and+identification+of+phosphorylation+sites.">Phosphopeptide mapping and identification of phosphorylation sites.</a> Curr Protoc Mol Biol. 18, Unit 18 19 (2001).</li><li>Doll, S., Burlingame, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+detection+and+assignment+of+protein+posttranslational+modifications.">Mass spectrometry-based detection and assignment of protein posttranslational modifications.</a> ACS chem. 10, 63-71 (2015).</li><li>Sharma, K., 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=Ultradeep+human+phosphoproteome+reveals+a+distinct+regulatory+nature+of+Tyr+and+Ser/Thr-based+signaling.">Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.</a> Cell rep. 8, 1583-1594 (2014).</li><li>Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+protein+function+by+multisite+phosphorylation--a+25+year+update.">The regulation of protein function by multisite phosphorylation--a 25 year update.</a> Trends Biochem Sci. 25, 596-601 (2000).</li><li>Walsh, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Posttranslation Modification of Proteins: Expanding Nature's Inventory. , (2006).</li><li>Boersema, P. 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=In-depth+qualitative+and+quantitative+profiling+of+tyrosine+phosphorylation+using+a+combination+of+phosphopeptide+immunoaffinity+purification+and+stable+isotope+dimethyl+labeling.">In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.</a> Mol Cell Proteomics. 9, 84-99 (2010).</li><li>Hutti, 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=A+rapid+method+for+determining+protein+kinase+phosphorylation+specificity.">A rapid method for determining protein kinase phosphorylation specificity.</a> Nat Methods. 1, 27-29 (2004).</li><li>Johnson, S. A., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinomics:+methods+for+deciphering+the+kinome.">Kinomics: methods for deciphering the kinome.</a> Nat Methods. 2, 17-25 (2005).</li><li>Pawson, T., Nash, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+cell+regulatory+systems+through+protein+interaction+domains.">Assembly of cell regulatory systems through protein interaction domains.</a> Science. 300, 445-452 (2003).</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=Analysis+of+yeast+protein+kinases+using+protein+chips.">Analysis of yeast protein kinases using protein chips.</a> Nat Genet. 26, 283-289 (2000).</li><li>Shah, K., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+genetic+approach+for+the+identification+of+direct+substrates+of+protein+kinases.">A chemical genetic approach for the identification of direct substrates of protein kinases.</a> Methods Mol Biol. 233, 253-271 (2003).</li><li>Zou, L., 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=PKIS:+computational+identification+of+protein+kinases+for+experimentally+discovered+protein+phosphorylation+sites.">PKIS: computational identification of protein kinases for experimentally discovered protein phosphorylation sites.</a> BMC bioinform. 14, 247 (2013).</li><li>Varjosalo, 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=Application+of+active+and+kinase-deficient+kinome+collection+for+identification+of+kinases+regulating+hedgehog+signaling.">Application of active and kinase-deficient kinome collection for identification of kinases regulating hedgehog signaling.</a> Cell. 133, 537-548 (2008).</li><li>Jansson, D., 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=Glucose+controls+CREB+activity+in+islet+cells+via+regulated+phosphorylation+of+TORC2.">Glucose controls CREB activity in islet cells via regulated phosphorylation of TORC2.</a> Proc Natl Acad Sci U S A. 105, 10161-10166 (2008).</li><li>Fu, A., Screaton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+kinomics+to+delineate+signaling+pathways:+control+of+CRTC2/TORC2+by+the+AMPK+family.">Using kinomics to delineate signaling pathways: control of CRTC2/TORC2 by the AMPK family.</a> Cell Cycle. 7, 3823-3828 (2008).</li><li>Abu-Thuraia, 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=Axl+phosphorylates+elmo+scaffold+proteins+to+promote+rac+activation+and+cell+invasion.">Axl phosphorylates elmo scaffold proteins to promote rac activation and cell invasion.</a> Mol Cell Biol. 35, 76-87 (2015).</li><li>Manning, G., Whyte, D. B., Martinez, R., Hunter, T., Sudarsanam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+kinase+complement+of+the+human+genome.">The protein kinase complement of the human genome.</a> Science. 298, 1912-1934 (2002).</li></ol>

The authors have nothing to disclose.

Depuis les publications originales décrivant l&#39;approche 14,15, la bibliothèque originale de 180 TPS-kinases a été élargi à 420 membres, soit environ 80% de la kinome protéine humaine. Avec la bibliothèque élargi, le protocole comme décrit prend 4-5 jours, puis 1-4 jours pour développer des films (le juge nécessaire), qui pourrait être raccourcie par l&#39;utilisation de phosphorimaging et l&#39;amélioration de signal numérique. Il ya plusieurs étapes clés où les soins doivent être prise...

Modifications post-traductionnelles de protéines (PTM) sont essentiels pour la communication intracellulaire. Peut-être le mieux étudié de PTM est tout phosphorylation, catalysée par la protéine-kinases, qui régulent une multitude de fonctions de protéines, y compris leur activité biochimique, la localisation subcellulaire, la conformation et la stabilité. L&#39;identification des sites de phosphorylation sur des protéines cibles peut être accomplie par la cartographie trypsique phosphopeptide ou par des techniques protéomiques maintenant standard en utilisant des échantillons enrichis pour des peptides phosphorylés 1,2. Alors que les trois quarts du protéome exprimé devraient être phosphorylée 3 et un identifiées 200.000 sites de phosphorylation 5, avec des estimations allant jusqu&#39;à 1 million 6, beaucoup d&#39;entre eux ont pas attribué la biologie, voie, ou la protéine kinase de signalisation. Bien que l&#39;identification des sites phosphorylés est relativement simple, comparativement plus grand défi est deidentifier la kinase apparenté (s) qui cible ces sites, un processus que nous appelons cartographie kinase: paires de substrat. Plusieurs approches pour l&#39;identification kinase: paires de substrats ont été décrits, que ce soit à partir d&#39;une kinase d&#39;intérêt et à la recherche de ses substrats ou à partir d&#39;un substrat d&#39;intérêt et d&#39;essayer de trouver une modification kinase expérimentalement ou par calcul 12 11.7. Pour identifier les kinases pour un substrat phosphorylé connu, la bioinformatique peuvent être utilisés pour identifier des protéines qui contiennent une séquence conservée courte d&#39;acides aminés flanquant le résidu phosphorylé (le site de consensus), ainsi que l&#39;identification des kinases qui forment un complexe précipitable avec le substrat. Cependant, ces approches sont longues et souvent ne répondent pas avec succès. Nous avons développé une approche fonctionnelle systématique pour identifier rapidement les kinases qui peut phosphoryler un substrat donné 13. Le test de l&#39;écran produit une excellente spécifiquelité, avec une sélection très clair pour les kinases apparentées potentiels. Compte tenu de la centralité de la phosphorylation de la signalisation biologique, l&#39;écran est utile pour la découverte dans la quasi-totalité des voies de signalisation cellulaires 14-16. L&#39;écran consiste à effectuer une analyse de kinase à grande échelle avec une bibliothèque de protéines kinases humaines. Les kinases ont été marqués avec du glutathion bactérienne S-transférase (GST) et sont purifiés à partir d&#39;extraits de cellules de mammifères, ce qui signifie que les enzymes recombinantes - à la différence de ceux qui sont préparés à partir de bactéries - sont générés en présence des protéines kinases en amont souvent requises pour la enzymes recombinantes ayant une activité in vitro. En effet, alors que l&#39;activité de kinase de sérine, de la thréonine, et la tyrosine requise pour l&#39;activation de la kinase en aval sont présents dans la levure 10, le génome de la levure codant pour 122 protéines kinases, ce qui indique que la kinome mammifère, plus de 500 gènes 17, est devenue nettement plus complexe dans le but de RegulaTe les processus uniques pour les organismes d&#39;ordre supérieur. De plus, l&#39;effet de différents stimuli pertinents pour la biologie cellulaire et la maladie humaine (tels que des petites molécules, facteurs de croissance, des hormones, etc.) peut être utilisé pour l&#39;activité de kinase de 14,15 moduler dans un contexte approprié.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lysis buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.1</td>
		</tr>
		<tr>
			<td>10x kinase buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.2</td>
		</tr>
		<tr>
			<td>10x M-ATP</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.3</td>
		</tr>
		<tr>
			<td>Human kinase plasmids</td>
			<td>Orfeome, Invitrogen, Origene</td>
			<td></td>
			<td>GST-tagged in house</td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Fisher Scientific</td>
			<td>CS003595</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Fisher Scientific</td>
			<td>SH3002201</td>
			<td>supplement with 100U/ml penicillin, 100ug/ml streptomycin, 10% fetal calf serum.</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture dishes</td>
			<td>Fisher Scientific</td>
			<td>877224</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Invitrogen</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated liquid dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>14072670</td>
			<td></td>
		</tr>
		<tr>
			<td>4mM pervanadate</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 3.1</td>
		</tr>
		<tr>
			<td>0.25 M CaCl2</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20-200 uL)</td>
			<td>Labnet</td>
			<td>p4812-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (1-10 uL)</td>
			<td>Thermo Scientific</td>
			<td>4661040</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 6-well plates</td>
			<td>Evergreen Scientific</td>
			<td>290-8116-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione coated 96-well plates</td>
			<td>Fisher Scientific</td>
			<td>PI-15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Biostad</td>
			<td>350355</td>
			<td></td>
		</tr>
		<tr>
			<td>GST tagged substrate</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP)</td>
			<td>Sigma</td>
			<td>M1891</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette (1 mL)</td>
			<td>Eppendorf</td>
			<td>22266209</td>
			<td></td>
		</tr>
		<tr>
			<td>32P gamma-ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU502Z500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>2X SDS lysis buffer (100 mL)</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.4</td>
		</tr>
		<tr>
			<td>26-well precast TGX gels</td>
			<td>BioRad</td>
			<td>567-1045</td>
			<td>gel percentage required is dependent on the molecular weight of the substrate of interest</td>
		</tr>
		<tr>
			<td>Coomassie stain</td>
			<td>Made in house</td>
			<td></td>
			<td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td>
		</tr>
		<tr>
			<td>Coomassie destain</td>
			<td>Made in house</td>
			<td></td>
			<td>10% acetic acid, 20% methanol</td>
		</tr>
		<tr>
			<td>Labeled gel containers</td>
			<td>Made in house</td>
			<td></td>
			<td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>Fisher Scientific</td>
			<td>57144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane sheets (2)</td>
			<td>BioRad</td>
			<td>165-0963</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Labconco</td>
			<td>4330150</td>
			<td></td>
		</tr>
		<tr>
			<td>Double emulsion autoradiography film</td>
			<td>VWR</td>
			<td>IB1651454</td>
			<td></td>
		</tr>
		<tr>
			<td>Film cassette</td>
			<td>Fisher Scientific</td>
			<td>FBAC-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Intensifying screen</td>
			<td>Fisher Scientific</td>
			<td>FBIS-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealing rubber roller</td>
			<td>Sigma</td>
			<td>R1275</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of kinase-substrate pairs using high throughput screening

Nous avons développé une plate-forme de criblage pour identifier kinases dédiés de protéines humaines pour les substrats phosphorylés qui peuvent être utilisés pour élucider de nouvelles voies de transduction du signal. Notre approche comprend l&#39;utilisation d&#39;une bibliothèque de protéines kinases humaine étiquetée GST purifiées et un substrat de protéine recombinante d&#39;intérêt. Nous avons utilisé cette technologie pour identifier MAP / microtubules affinité de régulation de la kinase 2 (MARK2) en tant que kinase pour un site de glucose régulé sur CREB-réglementées transcription coactivateur 2 (CRTC2), une protéine nécessaire pour la prolifération des cellules bêta, ainsi que la famille de tyrosine kinases de Axl tant que régulateurs de la métastase des cellules de la phosphorylation de la protéine adaptatrice rétroprojecteur. Nous décrivons cette technologie et nous discutons comment elle peut aider à établir une carte complète de la façon dont les cellules répondent aux stimuli environnementaux.

Des résultats représentatifs de l&#39;écran sont représentés sur la figure 2. 180 kinases ont été criblés en utilisant un peptide de substrat GST-marqué correspondant à aa 268-283 de CRTC2 ainsi que la protéine basique classique dosage de kinase substrat de la myéline (MBP). Seuls deux kinases, MARK2 et de la MARK3 de kinase hautement liés phosphorylés le peptide CRTC2. MBP est incluse en tant que contrôle interne dans tous les dosages, car il contient beaucoup de résidu...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identification de paires Kinase-substrat en utilisant criblage à haut débit


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

identification-kinase-substrate-pairs-using-high-throughput

Research

JoVE Journal

Biology

8.1K vues.  Children's Hospital of Eastern Ontario Research Institute. Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Vidéo: Identification of Kinase-substrate Pairs Using High Throughput Screening

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Criblage à haut débit et de biodétection avec fluorescent C. elegans Souches

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Recherche

Biologie

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Criblage à haut débit activité endoglucanase fongique dans les Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Haut débit cristallisation des protéines membranaires en utilisant la méthode lipidique Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

À haut débit de cartographie physique des chromosomes à l&#39;aide automatisée<em&gt; In situ</em&gt; Hybridation

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Criblage à haut débit pour les petites molécules modulateurs de canaux potassiques entrants Rectifier

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

ARN prédiction de la structure secondaire via SHAPE à haut débit

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Criblage à haut débit fonctionnelle utilisant une maison double lueur de dosage de luciférase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Automated Modular High Throughput exopolysaccharide plateforme de criblage Couplé avec très sensible Analyse Glucides d&#39;empreintes digitales

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multi-enzyme préalable au moyen d&#39;un système de criblage enzymatique génétique à haut débit

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...