C. elegans transgeni sono stati forniti dalla genetica Caenorhabditis Center (University of Minnesota, Minneapolis, MN). Questo lavoro è stato sostenuto da NIH R21 concedere NS0667678-01 a KS. Tutti gli autori hanno partecipato alla raccolta, analisi e interpretazione dei dati. CKL, KS, e KPC ha partecipato alla stesura e alla revisione del manoscritto.

1. Preparazione dei cibi verme batterica Giorno 1 <ol><li> Aggiungere 5 ml di saturi E. coli OP50 coltura batterica di 500 ml di brodo Terrific integrata con 50 mg / ml di streptomicina e crescere in un incubatore agitazione (225 rpm) durante la notte a 37 ° C. </li></ol> 2 ° giorno <ol start="2"><li> Suddividere la cultura batterica durante la notte in dieci provette da 50 ml e batteri centrifuga in una centrifuga refrigerata a 2.500 rcf per 20 minuti. </li><li> Decantare brodo LB e risospendere ogni pellet batterico in 10 ml di liquidi crescita nematode (NGM). Agitare orizzontalmente in un piano shaker per 15 minuti per risospendere i batteri. Per fare tampone NGM, aggiungere 3 g di NaCl a 1 L di acqua deionizzata e autoclave. Raffreddare a 55 ° C e aggiungere in ordine le seguenti soluzioni sterili: 1 ml di 1 M CaCl 2, 1 ml di 1 M MgSO 4, e 25 ml di 1 M di potassio fosfato, pH 6,0. </li><li> Centrifugare la coltura batterica in una centrifuga refrigerata a 2.500 rcf per 20 minuti. </li><li> Decantare il tampone in NGM e pesare il pellet batterico. </li><li> Aggiungere un volume equivalente di tampone NGM per risospendere il pellet, aliquota 3 ml di concentrato di batteri in provette da 15 ml, e conservare a -20 ° C. </li></ol> 2. Su larga scala C. elegans coltura liquida Noi usiamo una linea transgenica VP596 (dvIs19 [pAF15 (gst-4:: GFP:: NLS)]; vsIs33 [dop-3:: RFP]) portando due costrutti fluorescenti: Pgst -4:: GFP 14 a monitorare SKN-1 attività e PDOP-3:: RFP 17 a servire come standard per la normalizzazione verme numero. Giorno 1 <ol><li> Mescolare 150 ml NGM tampone, 1,5 ml di brodo LB, 150 ml di 5 mg / ml colesterolo (in etanolo), e 75 ml di 100 mg / ml di streptomicina. Filtro-sterilizzare la miscela e aggiungere in un matraccio 1 litro sterile. NOTA: L&#39;aggiunta di brodo LB 1% a tampone NGM aiuta a prevenire i vermi si attacchino al vetro e di plastica, ma questa somma non è sufficiente a sostenere la crescita dei batteri. </li><li> Sincronizzare i vermi con la procedura di ipoclorito di serie 18. Fino a 0,5 ml di vermi gravido possono essere elaborati in un unico tubo 15 ml con 5 ml di soluzione di ipoclorito (3,75 ml di acqua sterile, 1 ml di candeggina per uso domestico, e 250 microlitri 10 N NaOH). Lavare le uova rilasciato con acqua sterile e risospendere in 10 ml di tampone NGM. </li><li> Diluire un campione delle uova a 100 volte in NGM tampone (ad esempio, 100 l in 10 ml) e contare il numero di uova in tre distinte aliquote 5 microlitri. Multiplo del numero medio da 200.000 (100 fattore di diluizione x 2.000) per stimare il numero totale di uova. </li><li> Aggiungi 200.000 a 2 milioni di uova al pallone con il tampone NGM e agitare la cultura a 100 rpm a 20 ° C. </li></ol> 2 ° giorno <ol start="5"><li> Almeno 16 h dopo l&#39;aggiunta di uova al pallone, usare una pipetta sterile sierologico per rimuovere circa 0,5 ml di cultura verme sospeso. Pipettare 5 gocce tre microlitri sul coperchio sterile di una piastra di Petri. Mettere il coperchio in un congelatore di -20 ° C per 1-2 minuti per paralizzare i vermi. Contare il numero medio di vivere vermi nati per 5 microlitri e poi più di 30.000 (150 ml / 5 microlitri) per stimare il numero totale di vermi nati. </li><li> Scongelare un tubetto di surgelati OP50 coltura batterica (protocollo 1.6) e aggiungere 3 ml di 50% OP50 nella cultura verme vermi ogni 500.000 nati. Agitare il pallone a 100 rpm a 20 ° C. Worm si svilupperà in L4 e giovani adulti in circa 51 ore. L&#39;OD600 di batteri dovrebbe essere di circa 2,200 all&#39;inizio. Monitorare la quantità di batteri mediante assorbimento e aggiungere di più se il diametro esterno 600 scende al di sotto 0,900. </li></ol> 3. Worm raccolta e distribuzione 4 ° giorno <ol><li> Utilizzare una pipetta sterile sierologica per il trasferimento di circa 0,5 ml della cultura verme sospeso per una piastra standard agar NGM. Vedi i vermi con un stereomicroscopio per assicurarsi che la maggior parte sono L4 larve o giovani adulti. L4 avrà un posto ventrale chiara nel mezzo del corpo. I giovani adulti sarà leggermente più grande e non avrà un posto libero. </li><li> Versare la cultura verme in sterili provette da 50 ml e posto i tubi in un rack provetta sul bancone. Lasciare che il worm decantare per circa 10 minuti. Eliminare il surnatante tramite aspirazione o pipettaggio. NOTA: questo passaggio può essere importante per rimuovere le uova non schiuse o worm che non si è sviluppata perché si accontentano più lento di L4 e giovani adulti. </li><li> Raccogliere tutti i vermi in un unico tubo da 50 ml e lavare con tampone NGM con brodo LB 1% (NGM LB +) 3-4 volte a rimuovere i batteri. NOTA: Questo è fatto meglio in un secchio oscillante-centrifuga attraverso la raccolta di vermi a 500 rcf per 30 secondi e sostituendo il surnatante con freschi NGM + buffer di LB. </li><li> Dopo il lavaggio, riempire il tubo con NGM + tampone LB a 50 ml e versare in una bottiglia verme personalizzate di erogazione. Aggiungere un piccolo bar mescolare e mescolare per mantenere i vermi sospeso. Conte °indirizzo e numero di worm in tre 5 gocce microlitri come sopra indicato. Diluire con LB NGM + fino ad ottenere 12-15 worm per 5 goccia microlitri (~ 2,5-3,0 worm / mL). NOTA: un minimo di circa 20 ml (60.000 worm) è necessaria per riempire lo spazio morto del pallone di erogazione e cassetta dispenser. Ogni 384 pozzetti richiede circa 35.000 worm o 11,5 ml di vermi sospeso. </li><li> Carico di 10 microlitri cassetta di erogazione e sterilizzare in fondo con etanolo al 70%. Sciacquare l&#39;etanolo da fondo con acqua sterile. </li><li> Inserire l&#39;estremità della cassetta di erogazione nel pallone in modo che sia appena sopra la barra di mescolare senza interrompere il suo movimento. Assicurarsi che il worm rimane sospeso per tutto il fiasco intero. Programmare il dispenser per erogare a bassa velocità con 2 pre-impulsi. Primo ed eseguire almeno 10 ml di vermi sospeso. Riempire le piastre in base alle esigenze rapidamente per evitare che i vermi da stabilirsi nel tubo. </li><li> Sigillare i piatti con del nastro adesivo traspirante. </li><li> Posizionare le piastre su una piattaforma di agitazione in un incubatore alla temperatura appropriata per la propria analisi. Non impilare i piatti. </li></ol> 4. Analisi di fluorescenza <ol><li> Posizionare la piastra segnale in un lettore di micropiastre per misurare l&#39;intensità di fluorescenza di ogni bene con la lunghezza d&#39;onda appropriata di emissione e di eccitazione (Filtri per i nostri test: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em). </li><li> Calcolare il rapporto di GFP / RFP e normalizzare con le letture dei pozzi di controllo (senza attivare composto) per determinare la differenza esatta piega l&#39;intensità di fluorescenza derivati ​​da trattamenti individuali. </li></ol> 5. Rappresentante dei risultati: Il nostro SKN-1 test utilizza un ceppo cromosomicamente integrato giornalista doppio (VP596). Come mostrato nella Figura 1, il numero di worm è ben correlata al volume erogato. Come mostrato nelle figure 2A e 2B, totale GFP e RFP fluorescenza per bene è altamente riproducibile da pozzetto a pozzetto su una piastra ben 384. Come mostrato nella Figura 2C, non-indotta Pgst-4:: GFP fluorescenza è linearmente correlata alla PDOP-3:: RFP attraverso 384 pozzetti. Se espresso come rapporto di GFP / RFP, fluorescenza diventa altamente riproducibili da pozzetto a pozzetto attraverso una piastra ben 384 (confrontare coefficiente di variazione dalla figura 2D a 2A) che dimostrano la capacità del PDOP-3:: reporter RFP per ridurre la variabilità. Come mostrato nella Figura 3, l&#39;induzione di rispetto GFP a RFP con un SKN-1 attivazione xenobiotici (38 mM juglone) è robusto e altamente riproducibile su un piatto ben 384. <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="Figura 1"/> Figura 1. Numero di vermi rispetto al volume erogato. I worm sono stati diluiti a circa 2/μl e dispensati in un piatto ben 24 per il conteggio manuale con uno stereomicroscopio. N = 8 pozzetti per volume. <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="Figura 2"/> Figura 2. Fluorescenza totale di vermi dispensato in un piatto ben 384 è riproducibile. I worm sono stati diluiti a circa microlitri 2.5/μl e 30 è stato dispensato in tutti i pozzetti di una piastra ben 384. GFP (A) e RFP (B) fluorescenza di tutti i pozzetti aveva un coefficiente di variazione inferiore al 9%. (C) GFP fluorescenza è fortemente correlato alla RFP fluorescenza. (D) calcolo del rapporto tra GFP di RFP ridotto il coefficiente di variazione al di sotto del 6% (A, B e D) Le linee continue indicano i mezzi e le linee tratteggiate indicano tre deviazioni standard al di sopra o al di sotto della media. <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="Figura 3"/> . Figura 3 Attivazione di Pgst-4:: GFP è robusto e coerente. Circa il 75 L4 worm sono state erogate in tutti i pozzetti di una piastra ben 384 e 38 mM juglone è stato aggiunto in ogni colonna. GFP e RFP fluorescenza è stata misurata dopo 21 h di incubazione. La media del rapporto relativo fluorescenza di ogni controllo (1,0) e juglone pozzi (8,9) sono contrassegnati da linee continue. Tre deviazioni standard sopra la media dei controlli e al di sotto della media juglone sono segnati con linee spezzate.

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Nessun conflitto di interessi dichiarati.

Vi presentiamo un metodo per la coltura e l&#39;erogazione un gran numero di nematodi transgenici. Le attrezzature utilizzate per la coltura worm è standard per i laboratori che effettuano la clonazione molecolare e la gestione dei liquidi e delle attrezzature fluorescenza è standard per i laboratori di lavorazione un gran numero di micropiastre. Altri metodi di dispensare un gran numero di vivere C. elegans richiedono costosi impianti di smistamento delle particelle 19. Il Pgst-4:: GFP te...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> LB brodo </td><td> Ricerca Products International Corp. </td><td> L24066 </td><td></td></tr><tr><td> Terrific brodo </td><td> Ricerca Products International Corp. </td><td> T15100 </td><td></td></tr><tr><td> Synergy HT ™ Multi-mode micropiastre Reader </td><td> Biotek </td><td></td><td> Filtri: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em </td></tr><tr><td> MicroFlo Seleziona Dispenser </td><td> Biotek </td><td></td><td></td></tr><tr><td> Worm fiasco di erogazione </td><td> Southern Scientific Inc., Micanopy, FL </td><td></td><td> Assemblati su misura (352) 284-2531 </td></tr><tr><td> 384 micropiastre </td><td> Greiner Bio-One </td><td> 5678-1209 </td><td></td></tr><tr><td> Nastro di tenuta traspirante </td><td> Nunc </td><td> 241205 </td><td></td></tr><tr><td> (5-idrossi-p-naphthoqinone) Juglone </td><td> ACROS </td><td> 121640010 </td><td> Juglone è dissolto in DMSO </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D-5879 </td><td></td></tr><tr><td> C. elegans ceppo transgenico </td><td> Autore di laboratorio </td><td> VP596 </td><td> Pgst-4:: GFP e PDOP-3:: RFP </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

High-throughput screening (HTS) è un approccio potente per identificare modulatori chimici dei processi biologici. Tuttavia, molti composti identificati nel schermi utilizzando modelli di coltura cellulare si trovano spesso ad essere tossici o farmacologicamente attive in vivo 1-2. Lo screening in modelli animali tutto può aiutare ad evitare queste trappole e semplificare il percorso per lo sviluppo di farmaci. C. elegans è un organismo pluricellulare modello adatto per HTS. Si tratta di piccole dimensioni (&lt;1 mm) e può essere economicamente colto e dispensati in liquidi. C. elegans è anche uno dei modelli animali più docili sperimentalmente che consentono la determinazione rapida e dettagliata dei farmaci modalità d&#39;azione 3. Abbiamo descritto un protocollo per la coltura e l&#39;erogazione ceppi fluorescenti di C. elegans per high-throughput screening di librerie chimiche o rilevamento di contaminanti ambientali che alterano l&#39;espressione di un gene specifico. Un gran numero di vermi evolutivamente sincronizzati sono cresciuti in coltura liquida, raccolte, lavate, e sospeso ad una densità definita. I worm sono poi aggiunti al nero, dal fondo piatto a 384 pozzetti utilizzando un dosatore peristaltico liquido. Piccole molecole da una biblioteca chimica o campioni (per esempio, acqua, cibo, o suolo) possono essere aggiunti ai pozzetti con i vermi. In vivo, in tempo reale intensità di fluorescenza viene misurata con un lettore di fluorescenza per micropiastre. Questo metodo può essere adattato a qualsiasi gene inducibile in C. elegans per i quali un giornalista idonei. Lo stress inducibile molti e di sviluppo percorsi trascrizionale sono ben definiti in C. elegans GFP e reporter di ceppi transgenici esistono già per molti di loro 4. Quando combinato con i giornalisti appropriato transgenico, il nostro metodo può essere usato per individuare modulatori percorso o per sviluppare robuste analisi biosensore per i contaminanti ambientali. Dimostriamo il nostro C. elegans, la cultura e la dispensazione di protocollo con un dosaggio HTS abbiamo sviluppato per monitorare la C. trascrizione elegans cap &#39;n&#39; fattore collare SKN-1. SKN-1 e il suo omologo di mammifero Nrf2 attivare i geni citoprotettivo durante lo stress ossidativo e xenobiotici 5-10. Nrf2 mammiferi protegge da numerose malattie legate all&#39;età come il cancro, neurodegenerazione, e l&#39;infiammazione cronica ed è diventato uno dei principali bersagli chemioterapici 11-13. La nostra analisi si basa su un reporter GFP transgenico per il SKN-1 gene bersaglio -4 gst 14, che codifica per una glutatione s-transferasi 6. Il reporter gst -4 è anche un biosensore per le sostanze chimiche xenobiotiche e ossidativo che attivano SKN-1 e può essere utilizzato per rilevare i bassi livelli di contaminanti quali acrilamide e il metilmercurio 15-16.

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

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

Una procedura per liquidi a base di coltura e distribuzione dei<em&gt; C. elegans</em&gt; Giornalista ceppi che esprimono proteine ​​fluorescenti è descritto che non richiede costose attrezzature di smistamento. Questo approccio può essere applicato a numerosi inducibile<em&gt; C. elegans</em&gt; Geni per la scoperta di nuovi farmaci o sensori biologici di contaminanti.

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 ...

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

17.8K Views.  University of Florida. Una procedura per liquidi a base di coltura e distribuzione dei C. elegans Giornalista ceppi che esprimono proteine ​​fluorescenti è descritto che non richiede costose attrezzature di smistamento. Questo approccio può essere applicato a numerosi inducibile C. elegans Geni per la scoperta di nuovi farmaci o sensori biologici di contaminanti.

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

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 ...

Ricerca

Biologia

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi di screening per identificare fenotipi postembrionale in C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitative e automatica High-throughput Genome-wide Schermi RNAi in C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

High-throughput Purificazione di affinità-tagged proteine ​​ricombinanti

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

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, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans Chemiotassi Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochimica e High Throughput valutazione microscopica di massa grassa in<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

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 ...

Identification of Kinase-substrate Pairs Using 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 signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

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 ...