Questo lavoro è stato sostenuto da NSERC concessione 386634. Vorremmo ringraziare i membri della Screaton Lab per le discussioni utili.

1. Preparazione dei reagenti, piatti, e celle <ol><li> Rendere 500 ml di tampone di lisi: 25 mM Tris pH 7,5, NaCl 150 mM, 50 mM NaF, 0,5 mM EDTA pH 8.0, 0.5% TritonX-100, 5 mM beta-glicerofosfato, glicerolo 5%. Conservare a 4 ° C. Immediatamente prima dell&#39;uso, aggiungere 1 mM ditiotreitolo (DTT), 1 mM fenilmetil sulfonil fluoruro (PMSF), e 1 mM vanadato di sodio. Dopo questo passo, PMSF non è richiesto in ogni buffer di risciacquo. </li><li> Fai 20 ml di 10x Kinase Buffer: Tris 200 mM pH 7.5, 50 mM beta-glicerofosfato (FW 216), 2 mM vanadato di sodio. Aliquota in 1 ml provette e conservare a -20 ° C. Immediatamente prima dell&#39;uso, aggiungere 5 mM DTT. </li><li> Fai 20 ml di 10x tampone di M-ATP: 300 micron adenosina trifosfato (ATP), 66 mM MgCl 2, 33 MnCl mM 2. Aliquotare in 1 ml provette e conservare a -20 ° C. </li><li> Rendere 100 ml di 2x sodio dodecil solfato (SDS) tampone di lisi: 1,5 g base di TRIS, 20 ml di glicerolo, 30 ml di H 2 O. Sciogliere e regolare il pH a 6,8 con HCl. Aggiungere 40ml 10% SDS, regolare il volume a 100 ml. Aggiungere 25 mg di blu di bromofenolo. </li><li> Spot 4 ml di 25 ng / ml plasmide di espressione di mammifero che codificano un GST-chinasi 14 per bene in gruppi di 96 pozzetti e targhette di conseguenza. Seal e congelare le piastre a -20 ° C fino al momento dell&#39;uso. </li><li> 1-2 giorni prima della trasfezione, le cellule cultura HEK293T in Modified Eagle Medium completo di Dulbecco (DMEM) + 10% siero fetale bovino (FBS) e antibiotici in un 37 ° C incubatore integrato con CO 2 (finale 5%). Passaggio con tripsina e non consentono lo stock di diventare&gt; 80% confluenti durante l&#39;espansione. Un minimo di 6 x 10 7 cellule sono necessari per lo schermo (circa 3 x 15 cm piatti di cellule HEK293T al 80% confluenza). </li></ol> 2. Trasfezione Nota: Vedere la Figura 1 per un diagramma di flusso intero protocollo. <ol><li> Se sono stati congelati piastre contenenti plasmidi chinasi, scongelare a temperatura ambiente unnd centrifugare a 1900 xg per 3 minuti per raccogliere l&#39;umidità al fondo dei pozzetti. </li><li> Mescolare 8,6 ml di mezzo di siero ridotta (ad esempio, OPTI-MEM) con 312,7 ml di base lipidica trasfezione reagente. Lasciate riposare per 5 minuti. </li><li> Aggiungere 10 ml di mezzo di siero ridotto a ciascun pozzetto usando un dosatore automatico provvisto di un piccolo volume di cassette. </li><li> Aggiungere 10 ml per pozzetto di riduzione a medio siero / trasfezione miscela reagente dal punto 2.2 utilizzando un dosatore automatico provvisto di un piccolo vassoio di volume. Lasciate riposare per 20 a 45 minuti. </li><li> Risospendere le cellule 293T a 7,5 x 10 5 cellule / ml a 80 ml ​​di DMEM completo. Aggiungere 100 ml di sospensione cellulare (cioè 7,5 x 10 4 cellule) per pozzetto utilizzando un dosatore automatico munito di una cassetta volume standard. </li><li> Controllare pozzi al microscopio per la distribuzione cellulare uniforme e tornare a incubatore per 24 ore. </li></ol> Pulldown 3. GST-chinasi <ol><li> Fai fresh: 4 mm soluzione pervanadate mescolando 60 ml di 0,2 M vanadato di sodio con 540 ml di H 2 O. In una seconda miscela tubo 2,7 ml di perossido di 30% e 1,4 ml di PBS. Aggiungere le due soluzioni insieme e lasciate riposare per 15 minuti prima dell&#39;uso. </li><li> Usando una pipetta multicanale (non utilizzare un distributore automatico di liquido in quanto crea troppa turbolenza nel pozzo) dispensare 2 ml di 0,25 M CaCl 2 in ogni pozzetto, seguiti da 2,5 ml di pervanadate preparato al punto 3.1. Incubare ogni piastra a 37 ° C per 10 min e poi posto su ghiaccio. </li><li> Preparare 35 ml di tampone di lisi con l&#39;aggiunta di DTT, PMSF e vanadato di sodio come indicato nella Sezione 1 (Preparazione dei reagenti). </li><li> Mantenere le piastre in ghiaccio, rimuovere media da ogni pozzetto usando un aspiratore a vuoto. Aggiungere immediatamente 50 ml / pozzetto di tampone di lisi freddo ghiaccio utilizzando un dosatore automatico munito di una cassetta standard. Lasciare riposare 30 minuti in ghiaccio per Lyse (opzionale: può fermare qui, se necessario, plat di tenutaE e memorizzazione a -80 ° C. Per continuare, piastre disgelo su ghiaccio). </li><li> Piastre far girare a 1900 xg per 3 minuti a 4 ° C. </li><li> Raschiare le cellule da ciascun pozzetto utilizzando una pipetta multicanale e trasferire tutti i contenuti per etichettato in modo appropriato piastre 96 e V-bottom. Piastre far girare a 1900 xg per 10 minuti a 4 ° C. </li><li> Durante la rotazione, riempire piastre rivestite glutatione (uno per ogni piastra a 96 pozzetti) con 100 l / pozzetto di ghiaccio tampone di lisi freddo (senza PMSF) come un risciacquo. Mantenere le piastre in ghiaccio. </li><li> Rimuovere piastre V-bottom dalla centrifuga. Uno alla volta, capovolgere le piastre di glutatione su un lavandino per scuotere il tampone di lisi e macchia su un tovagliolo di carta. Trasferire tampone di lisi dalle piastre V-bottom alle piastre glutatione inclinando la piastra e con una pipetta multicanale, facendo attenzione a non disturbare il pellet sul fondo. Coprire e lasciare le piastre in ghiaccio per un minimo di 2 ore per legare. </li><li> Vicino alla fine della fase di legame 2 hr, preparare una workstation radioattività, garantendo tegli necessarie precauzioni di sicurezza sono in atto per il lavoro radioattivo. Impostare fornetto di 30 ° C. </li><li> Preparare 200 ml di tampone di lisi aggiungendo DTT e sodio vanadate come indicato nella Sezione 1. PMSF non è necessario in questa fase. </li><li> Capovolgere le piastre di glutatione su un lavandino per scuotere il tampone di lisi e asciugare su un tovagliolo di carta. Sciacquare pozzi 3x con 100 microlitri tampone di lisi (senza PMSF). Non lasciate che i pozzi si siedono a secco - tenerli in risciacquo fino al momento di procedere. </li><li> Preparare 55 ml di tampone chinasi 1x (KB) diluendo lo stock 10x e l&#39;aggiunta di DTT come indicato nella Sezione 1. placche Sciacquare una volta con 50 ml di 1x KB utilizzando un dosatore automatico dotato di una cassetta volume standard. Lasciare 1x KB in pozzi fino a quando la soluzione A è pronta: <ol><li> Preparare una soluzione aggiungendo 500 a 530 ug del substrato di interesse, 500 mg di proteina basica della mielina (MBP), 2,65 ml di 10x KB, 13.25 ml di 1 M DTT e H 2 O fino a 15,9 ml. </li></ol></li&gt; <li> Uno alla volta, capovolgere le piastre su un lavandino per rimuovere 1x KB risciacquo, asciugare su carta assorbente, e subito aggiungere 30 ml di soluzione A utilizzando un dosatore automatico provvisto di un piccolo volume di cassette. Mantenere le piastre in ghiaccio. </li><li> Preparare Soluzione B nell&#39;area di lavoro radioattività aggiungendo 2,5 ml 10x M-ATP, 500 pCi gamma- 32 P ATP, e H 2 O a 10 ml. <ol><li> Aggiungere 20 ml di soluzione B per bene con una pipetta repeater che aiuta nel mescolare dovuto alla forza di espulsione. Coprire e incubare a 30 ° C ibridazione forno per 30 min. </li></ol></li><li> Dopo 30 minuti, trasferire lastre di nuovo al ghiaccio. Aggiungere 50 ml di tampone di lisi 2x SDS in ogni pozzetto usando una pipetta multicanale. Può procedere a questo punto il passo successivo, o sigillare le piastre con un foglio di alluminio e conservare a -20 ° C fino conveniente. </li></ol> 4. Esecuzione, colorazione, e gel di essiccazione Nota: Tutti i lavori devono essere eseguiti in una zona DESIGNAted per la radioattività. <ol><li> Accendete il forno di ibridazione e impostato a 85 ° C. Piastre scongelare a temperatura ambiente. Una volta che il forno ha raggiunto la temperatura, piastre di trasferimento a forno e incubare per 10 minuti a denaturare campioni. </li><li> Carico di 26 e gel prefabbricati con 15 ml di ogni reazione utilizzando una pipetta multicanale per riempire diversi pozzi in una sola volta. Bisogna fare attenzione che tutti i suggerimenti siano allineati con le corrispondenti bene prima di aggiungere i campioni. Eseguire gel a 150 V. Non lasciate che il colorante inseguimento (linea blu) scappare del fondo del gel come questo contiene il prive di personalità giuridica ATP. </li><li> Smontare i gel e tagliare la prive di personalità giuridica ATP (linea blu) con un bisturi o un bordo dritto come sarà sovraesporre i film. Mettere il gel in contenitori etichettati e coprire con macchia Coomassie per 15 min. </li><li> Rimuovere la macchia Coomassie, sciacquare brevemente il gel con acqua e aggiungere Decolorare soluzione. Decolorare i gel finché le proteine ​​sono chiaramente visibili. Una band per MBP e una banda per il substrato dovrebbevisibile per ogni campione. </li><li> Per asciugare i gel: <ol><li> Tagliare un grande foglio di carta da filtro e posizionarlo sul asciugatrice. </li><li> Bagnate un foglio di cellophane in acqua distillata fino a che non è liscia e ruga libero, e posizionarlo sulla parte superiore del foglio. </li><li> Disporre il gel sulla parte superiore del foglio di cellophane, prendendo nota dell&#39;ordine di gel. Bagnare un secondo foglio di cellophane e posto sulla sommità del gel. </li><li> Stendere tutte le bolle (un rullo di tenuta piatto funziona bene per questo) per una superficie bella uniforme. Chiudere il lembo, attivare il vuoto, ed asciugare il gel per 3 ore a 80 ° C. </li></ol></li><li> Una volta che i gel sono asciutti, esporli a pellicola XAR utilizzando uno schermo per intensificare il segnale. Avvolgere la cassetta con involucro di saran o un sacchetto di plastica e sigillare con nastro adesivo per tenere fuori gelo. Conservare la cassetta a -80 ° C durante la notte. </li></ol> 5. Sviluppare XAR Films <ol><li> Il giorno seguente, rimuovere cassetta dal freezer e lasciare scongelare a temperatura ambiente. Sviluppare il film inuna camera oscura utilizzando un processore pellicola secondo le istruzioni del produttore. Nota: Esaminare i film per la prova di coppie chinasi-substrato. Una seconda esposizione più lunga può anche essere utile per rilevare eventi di fosforilazione più deboli. </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.

Poiché le pubblicazioni originali descrivono l&#39;approccio 14,15, la libreria originale di 180 GST-chinasi è stata ampliata per 420 membri, o ~ 80% della proteina kinome umana. Con la libreria espanso, il protocollo come descritto richiede 4-5 giorni e poi 1-4 giorni per sviluppare film (se ritenuto necessario), che può essere ridotto mediante l&#39;uso di phosphorimaging e valorizzazione segnale digitale. Ci sono diversi passaggi chiave in cui occorre prendere cura (vedi figura 1 per un...

Proteina modificazioni post-traduzionali (PTM) sono essenziali per la comunicazione intracellulare. Forse il più studiato di tutti PTM è fosforilazione, catalizzata da proteina chinasi, che regolano una miriade di funzioni proteiche, compresa la loro attività biochimica, localizzazione subcellulare, conformazione e stabilità. L&#39;individuazione dei siti di fosforilazione di proteine ​​bersaglio può essere eseguito la mappatura fosfopeptide trittico o mediante tecniche di proteomica ormai standard che utilizzano i campioni arricchito da peptidi fosforilati 1,2. Mentre tre quarti del proteoma espresso dovrebbero essere fosforilata 3 e identificate 200.000 siti di fosforilazione 5, con stime fino a 1 milione 6, molti di questi non hanno biologia assegnato, percorso di segnalazione, o proteine ​​chinasi. Mentre individuazione dei siti fosforilazione è relativamente semplice, una sfida comparativamente più grande è quella diidentificare la chinasi affine (s) che gli obiettivi di questi siti, un processo che chiamiamo mappatura chinasi: coppie di substrato. Diversi approcci per identificare chinasi: coppie di substrato sono stati descritti, sia partendo con una chinasi di interesse e cerchi suoi substrati o iniziare con un substrato di interesse e il tentativo di trovare una chinasi modificante sperimentalmente 7-11 o computazionalmente 12. Per identificare chinasi per un noto substrato fosforilato, bioinformatica possono essere utilizzate per identificare le proteine ​​che contengono una breve sequenza di amminoacidi conservati che fiancheggiano il residuo fosforilato (sito consensus), nonché individuare chinasi che formano un complesso precipitable con il substrato. Tuttavia, questi approcci sono in termini di tempo e spesso non hanno successo. Abbiamo sviluppato un approccio funzionale sistematica di individuare rapidamente chinasi che può fosforilare un determinato substrato 13. Il saggio schermo produce eccellente specificolità, con molto chiara selezione di potenziali chinasi affini. Data la centralità della fosforilazione di segnalazione biologica, lo schermo è utile per la scoperta in quasi tutti i cellulari vie di segnalazione 14-16. Lo schermo comporta l&#39;esecuzione di un test chinasi larga scala con una libreria di proteine ​​chinasi umani. Le chinasi sono state contrassegnate con glutatione batterica S-transferasi (GST) proteine ​​e sono purificato da estratti cellulari di mammifero, il che significa che gli enzimi ricombinanti - a differenza di quelli preparati da batteri - sono generati in presenza delle proteine ​​chinasi a monte spesso necessari per la enzimi ricombinanti hanno attività in vitro. Infatti, mentre l&#39;attività della chinasi serina, treonina e tirosina per l&#39;attivazione della chinasi a valle sono presenti nel lievito 10, il genoma di lievito codifica 122 protein chinasi, indicando che il kinome mammiferi, con oltre 500 geni 17, è diventato molto più complesso per regulaTE i processi unici per gli organismi di ordine superiore. Inoltre, l&#39;effetto di diversi stimoli rilevanti alla biologia cellulare e malattie umane (quali piccole molecole, fattori di crescita, ormoni, ecc) può essere utilizzato per attività chinasica 14,15 modulate in un contesto appropriato.

<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

Abbiamo sviluppato una piattaforma di screening per identificare proteine ​​chinasi dedicati umani per substrati fosforilati che possono essere utilizzati per delucidare nuovi percorsi di trasduzione del segnale. Il nostro approccio è caratterizzato dall&#39;uso di una libreria di purificate protein chinasi umana GST-tag e una proteina ricombinante di interesse substrato. Abbiamo utilizzato questa tecnologia per identificare MAP / microtubuli affinità regolazione chinasi 2 (MARK2) come chinasi per un sito di glucosio-espresso nelle CREB-regolato trascrizionale coattivatore 2 (CRTC2), una proteina necessaria per la proliferazione delle cellule beta, nonché la famiglia di tirosina chinasi Axl come regolatori di metastasi delle cellule di fosforilazione della proteina dell&#39;adattatore ELMO. Descriviamo questa tecnologia e discutiamo come può aiutare a stabilire una mappa completa di come le cellule rispondono agli stimoli ambientali.

I risultati rappresentativi di uno schermo sono illustrati nella Figura 2. 180 chinasi sono stati proiettati utilizzando un peptide substrato GST-tag corrispondenti a aa 268-283 da CRTC2 così come la proteina basica classico saggio di chinasi substrato mielina (MBP). Solo due chinasi, MARK2 e altamente connessi MARK3 chinasi fosforilate il peptide CRTC2. MBP è incluso come controllo interno in tutti i saggi, in quanto contiene molti residui fosforilabili e funziona a 18 kDa, verso il ...

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.

Identificazione delle coppie Kinase-substrato Usando High Throughput Screening


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 Views.  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.

Video: 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.

High-throughput screening e biopercezione con fluorescente C. elegans Ceppi

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

Ricerca

Biologia

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.

Throughput Screening elevati di attività Endoglucanase fungine in 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.

Cristallizzazione high-throughput di proteine ​​di membrana con il metodo lipidica 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.

High-throughput mappatura fisica dei cromosomi con modalità automatizzate<em&gt; In situ</em&gt; Ibridazione

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.

High-throughput screening per la piccola molecola modulatori di canali di potassio Inward 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.

RNA Secondaria previsione della struttura utilizzando la forma di alta velocità

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.

High-throughput screening funzionale utilizzando una casalinga 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 ...

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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.

Automatizzata Modular High Throughput Screening esopolisaccaride piattaforma Accoppiato con altamente sensibile Analisi Carboidrati impronte digitali

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-enzima screening utilizzando un high-throughput genetica 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 ...