Ringraziamo EUROSARF per averci inviato il lievito TAP plasmidi di espressione. Siamo anche grati per l&#39;aiuto editoriale da Ryan Labadens. Questo lavoro è stato sostenuto da una borsa NIH / NINDS (R01NS070962) per CW

1. Generare UAS-TAP-tag mosche transgeniche <ol><li> Generare pUAST-TAP-tagged costrutti di DNA. <ol><li> Decidere quale lato (N-o C-terminale) della proteina esca il tag TAP deve essere fuso a, sulla base della struttura / funzione della proteina. Si veda la discussione per ulteriori dettagli. </li><li> Subclone la regione codificante del cDNA del gene di interesse nei siti di clonazione multipla (MCS) dei vettori pUAST-NTAP o pUAST-CTAP per generare N-o C-terminale con targhetta transgeni UAS-TAP, rispettivamente. Vedere la Figura 1 per le mappe dettagliate e siti di restrizione utilizzabili e cornici di lettura. </li></ol></li><li> Generare UAS-TAP-tagged mosche transgeniche. <ol><li> Generare mosche transgeniche seguenti protocolli standard utilizzando P-elemento-mediata di inserimento 11. Un certo numero di servizi di iniezione sono disponibili in commercio. </li><li> Attraversare neuronale autista GAL4 (cioè BG380-GAL4) per ogni singola linea transgenica e determinare i livelli di espressione della proteinadi ogni riga mediante western blot (con anticorpi perossidasi anti-perossidasi) e / o immunostaining (con anticorpo anti-TAP). In generale, una linea transgenica con un livello di espressione della proteina che si trova vicino alla proteina endogena è raccomandato per procedure TAP. Si veda la discussione per ulteriori dettagli. </li><li> PerformGAL4/UAS-based esperimenti di salvataggio per confermare la funzionalità dei transgeni TAP-tag se sono disponibili la perdita di funzione mutanti dei geni di interesse. Scegliere un transgene che può salvare la sostanza dei fenotipi mutanti per i seguenti esperimenti TAP. </li></ol></li></ol> 2. Preparare i campioni per TAP procedura <ol><li> Generare un titolo mosca che porta sia un driver neuronale GAL4 (es. BG380-Gal4) e il transgene TAP-tag scelti in modo da facilitare l&#39;espansione dei campioni mosca. Raccogliere le progenie F1 del conducente GAL4 e la croce UAS-transgene nei rari casi in cui la combinazione di cui sopra determina la sopravvivenza e la crescita di svantaggio. &lt;/ Li&gt; <li> Raccogliere campioni di piccole dimensioni e ottimizzare le condizioni di lisi per solubilizzare la proteina TAP-tag. <ol><li> Effettuare una serie di buffer di lisi utilizzando una combinazione dei detergenti non ionici NP-40 (0,1-1%), NaDOC (0,1-1%) e Triton X-100 (0,05-0,5%). Vedi Tabella 1 e di discussione per maggiori informazioni. </li><li> Sulla cima di un pad CO2, utilizzare # 5 pinze per sezionare 20 capi adulti dal transgene TAP esprimere mosche e raccoglierli in una provetta da 1,5 ml a testare ogni condizione di buffer di lisi. </li><li> Aggiungere 100 ul di buffer test di lisi al tubo e omogeneizzare le testine segnando su e giù con un pestello in plastica, poi aggiungere un altro buffer test 100 ul. </li><li> Centrifugare il lisato testa a 21.500 xg per 10 min (4 ° C), e separare il surnatante e pellet dopo centrifugazione. Aggiungere 25 ul 2x SDS tampone di caricamento per il pellet e tampone di caricamento 10 ul 4x SDS a 30 ul di surnatante, rispettivamente. </li><li> Bollire i due campioni per 5 min e analizzarli side-byLato usando SDS-PAGE e successivo Western Blot con l&#39;anticorpo PAP. Determinare la solubilità dal rapporto tra livelli TAP-proteina nelle supernatante vs il pellet. </li></ol></li><li> Preparare campione in larga scala <ol><li> Espandere e raccogliere mosche adulte. <ol><li> Espandere lo stock neuronal-GAL4/UAS-TAP-transgene in bottiglie e capovolgere le bottiglie ogni 3 giorni fino cumulative di 250 bottiglie vengono utilizzati per la raccolta. </li><li> Raccogliere 1-3 giorni di età adulta vola in 50 ml provette coniche, mettere il tubo in azoto liquido immediatamente Deep Freeze linea. Conservare le mosche in una -80 ° C freezer. Si noti che il volume di linea non deve superare i 2/3 del tubo da 50 ml. </li></ol></li><li> Raccogliere teste fly (eseguire questo passaggio sulla cima di ghiaccio secco in polvere). <ol><li> Estrarre i setacci prechilled e il mortaio e pestello da -80 ° C freezer e metterli su ghiaccio secco, idealmente all&#39;interno di un grande secchio di ghiaccio. Stack due setacci di prova standard USA con un No. 25 su °e superiore e No. 40 in basso. </li><li> Prendere le mosche congelati fuori e farli cadere in azoto liquido e conservare in linea in là per circa 10 min. Vortex o agitare le provette energicamente per rompere le teste, le gambe e le ali dai corpi. </li><li> Versare il composto al setaccio superiore, e quindi scuotere i setacci energicamente tenendo setacci insieme. Dopo la setacciatura, i corpi rimarranno sul setaccio superiore, volare teste saranno conservati sul setaccio di fondo, e le gambe, ali, e altri detriti cadranno al ghiaccio secco. Separare i due setacci e con attenzione trasferire i capi mosca al mortaio freddo. </li></ol></li><li> Omogeneizzare le teste fly <ol><li> In cima a ghiaccio secco, macinare i capi con il mortaio e il pestello di particelle in polvere, poi trasferire la polvere a 15 ml di vetro Dounce Tissue Grinder che è stato prechilled sul ghiaccio. </li><li> Misurare i pesi della smerigliatrice prima e dopo il campione testa è stata versata in esso, e quindi calcolare qual è il samp testale pesa. Un totale di 6-15 grammi di teste fly sarà sufficiente per ciascun esperimento TAP regolando di conseguenza per i livelli di espressione della proteina. Aggiungere 15 ml di tampone di omogeneizzazione ghiacciato (tampone di lisi ottimizzata nel passaggio 2.2) per la polvere e poi corsa con il grande pestello pallone fino a che è facile per il pestello per andare su e giù. Mantenere la smerigliatrice vetro su ghiaccio in ogni momento. </li></ol></li><li> Preparare il surnatante per TAP <ol><li> Trasferire l&#39;omogeneizzato in una provetta da centrifuga ad alta velocità e centrifuga per 20 minuti a 50.000 xg ~ (4 ° C). Trasferire il surnatante in una nuova provetta da centrifuga ad alta velocità e ripetere la centrifugazione ancora una volta. </li><li> Trasferire il supernatante in un tubo ultracentrifuga ed eseguire un 40 min ~ 250.000 xg centrifuga per eliminare ulteriormente il surnatante. Il supernatante è pronto per le procedure di purificazione per affinità tandem dopo ultracentrifugazione. </li></ol></li></ol></li></ol> 3. TAP Purificazione </p&gt; Le sezioni che seguono sono stati ricavati dal laboratorio TAP Séraphin protocol 12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf ) <ol><li> Eseguire IgG tallone purificazione per affinità <ol><li> Preparare IgG Sepharose tallone mentre i campioni vengono centrifugati. Lavare 400 microlitri 3x perline IgG in un tubo Falcon da 15 ml con 10 ml di freddo tampone di lavaggio IgG. Per ogni lavaggio, scuotere delicatamente il tubo per 2 minuti, e poi girare le perle a 1.000 xg per altri 2 min. Alla fine del terzo lavaggio, rimuovere il tampone e lasciare solo le perline nel tubo. </li><li> Trasferire con cautela il surnatante eliminato (~ 15 ml) nel tubo da 15 ml contenente le perline IgG. Incubare le perline e mix lisato cervello a 4 º C su una nutator per 2 ore. </li><li> Impostare un micro colonna pulito e vuoto con circa 15 ml di volume totale nella stanza fredda. Caricare la miscela sferette IgG dalla costante versare la miscela nella colonna, non cercare di intrappolare qualsiasibolle d&#39;aria all&#39;interno della colonna. Lasciare le perle di stabilirsi nella colonna e il buffer per drenare lentamente flusso di gravità. </li><li> Lavare la colonna a fondo con 10 ml di freddo tampone di lavaggio IgG dopo tutto il lisato cervello ha fluito attraverso la colonna IgG risolta. Ripetere il lavaggio 2x. Nota: non lasciare mai che il tallone asciugare all&#39;aria. </li></ol></li><li> Eseguire TEV scissione <ol><li> Dopo il terzo lavaggio, lavare nuovamente la colonna con 10 ml di tampone TEV scissione. Questo passaggio prepara il tallone IgG che sequestranti il ​​complesso esca per TEV scissione. </li><li> Destra prima dell&#39;ultima goccia di tampone TEV sta per gocciolare, mettere un tappo nella parte inferiore della colonna di bloccare il flusso, aggiungere 1,3 ml di tampone TEV contenente 130 unità di enzima TEV alla colonna, e quindi tappare saldamente cima colonna. Assicurarsi che la colonna è sigillato bene ad entrambe le estremità. </li><li> Ruotare la colonna a 18 ° C per 2 ore per consentire l&#39;enzima TEV per fendere il peptide al sito TEV e rilasciare il complesso proteico WHIle lasciandosi alle spalle la proteina A peptide dominio legata alle perle del IgG Sepharose. </li></ol></li><li> Eseguire Calmodulina tallone purificazione per affinità <ol><li> Preparare le perline calmodulina mentre le perle IgG vengono incubate con l&#39;enzima TEV. Lavare 200 microlitri calmodulina perline in 15 ml di 3x tubo Falcon, ogni volta con 10 ml di freddo Calmodulin binding buffer. Per ogni lavaggio, scuotere delicatamente il tubo per 2 minuti su un nutator, e poi girare le perle a 1.000 xg per 2 minuti. Alla fine del terzo lavaggio, estrarre tutto il buffer e lasciare solo le perline nel tubo. </li><li> Al termine dell&#39;incubazione TEV (fase 3.2.3), riportare la colonna IgG torna alla camera fredda e impostare verso l&#39;alto. Lasciate le perline accontentarsi di 10 min. </li><li> Togliere il tappo superiore e poi il tappo inferiore, e poi raccogliere il 1.3 ml TEV prodotto di scissione in un tubo Falcon 15 ml. Lasciate che il buffer di scarico completamente. Aggiungere un ulteriore buffer di 200 microlitri TEV alla colonna per spingere fuori il volume morto della colonna, raccogliere la fbasso nello stesso tubo. </li><li> Aggiungere 4,5 ml di tampone di legame Calmodulin e 4,5 ml 1 M CaCl 2 per il prodotto di scissione 1,5 ml TEV raccolti sopra. Il CaCl 2 serve per titolare l&#39;EDTA nel buffer TEV. Trasferire la miscela 6 ml nella provetta contenente le perline calmodulina e ruotare il tubo a 4 ° C su una nutator per 1 ora. </li><li> Impostare un altro micro colonna pulito e vuoto con circa 10 ml di volume totale nella stanza fredda. Caricare la miscela sferette Calmodulina alla colonna e lasciarlo scolare per gravità. </li><li> Quando tutta la soluzione è fluito attraverso la colonna Calmodulina costante, lavare la colonna due volte, ciascuna con 10 ml di freddo Calmodulin binding buffer. Nota: evitare di disturbare le perline calmodulina e cercare di mantenere la superficie delle perle più piatta possibile durante il lavaggio. </li></ol></li><li> Eluire il complesso esca dalla colonna Calmodulin. Subito dopo il lavaggio, eluire la colonna Calmodulina con cinque frazioni di 200 microlitri freddo Calmodulin tampone di eluizione. Per ciascuna frazione, aggiungere lentamente 200 ml di tampone di eluizione alla colonna e raccogliere l&#39;eluato con una marcata 1,5 ml provetta Eppendorf. Ripetere questa 4x. </li><li> Analizzare il complesso proteico mediante SDS-PAGE <ol><li> Prendere una piccola aliquota da ciascuna delle cinque frazioni (circa 30 mL) e aggiungere tampone di caricamento SDS. Bollire i campioni per 5 min e caricare i campioni side-by-side con marcatori molecolari di proteine ​​in un gradiente (4-15%) SDS-PAGE gel. </li><li> Dopo che i campioni sono completamente risolti nel gel, fermare la elettroforesi e macchiare il gel con qualsiasi sensibili colloidali procedure di colorazione Coomassie basate G-250 come &#39;argento blu&#39; colorazione 13. Argento colorazione è opzionale ma non preferibile perché non è completamente compatibile con la successiva analisi di spettrometria di massa. Conservare il resto l&#39;eluato in un congelatore -80 ° C per ulteriori analisi quali spettrometria di massa per scoprire le identità molecolare del complesso proteina purificata. See discussione. </li></ol></li></ol>

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Metodo Tandem purificazione per affinità (TAP) offre un protocollo dual purificazione che permette l&#39;isolamento e l&#39;arricchimento di complessi proteici attraverso due passaggi di purificazione di affinità indipendenti. Il design del tag TAP non è limitata a ciò che viene presentato in questo protocollo, le altre proteine ​​domini di legame e motivi sono applicabili anche se le condizioni del buffer vengono adeguati di conseguenza. Un buon esempio di altri tag TAP è il tag GS-TAP, una combinazione di una...

Definizione dei meccanismi molecolari o reti che mediano un particolare processo biologico è uno degli obiettivi finali della ricerca biomedica. Fly genetisti hanno dipendeva pesantemente sulla genetica in avanti, soprattutto modificatore schermi genetici (sia enhancer e schermi soppressori), per identificare i fattori che lavorano insieme, in parallelo, o monte oa valle di un gene di interesse. Tuttavia, gli schermi genetica in avanti spesso non riescono a identificare i geni essenziali che, se mutato, causa letalità nelle fasi precoci dello sviluppo, oi geni con ridondanza funzionale e compensazione la cui perdita di funzione solo causare difetti sottili che sono difficili da segnare. Un modo per superare questa difficoltà è quello di schermo per interazioni dirette proteina-proteina. Per più di un decennio, un crescente elenco di metodi biochimici, tra cui due ibridi di lievito, esposizione su fago, reticolazione chimica, Co-IP, Tandem Affinity Purification (TAP), etc. sono stati utilizzati per studiare proteine-proteina interazioni. Ognuno di questi approcci ha una propria serie di punti di forza e di debolezza in materia di sensibilità e specificità. Tra questi, il metodo TAP consente il rilevamento di interazione fisica in condizioni quasi fisiologiche, conserva specificità e consistenza 2 e include la possibilità di estendere a high-throughput analisi 3,4. Il metodo TAP è stato originariamente sviluppato nel lievito da colleghi Rigautand 1. In questo metodo, una proteina di interesse è espressa con un tag TAP. Il tag TAP ospita due affinità di legame domini indipendenti: una proteina Un dominio che si lega a IgG e un dominio-calmodulina vincolante. I due domini sono separati da una TEV (Tobacco Etch Virus) sito di clivaggio. Tale combinazione permette per due turni indipendenti di purificazioni affinità per ridurre sufficientemente le associazioni aspecifici e arricchiscono le associazioni specifiche 1. Per questo esempio, il metodo TAP è un potente metodologiad identificare interazioni in vivo di una data proteina, sebbene sovraesprimono la proteina esogena può rendere più inclini ad associare proteine ​​che normalmente non complesso con la sua controparte endogena. Dal suo sviluppo, il metodo TAP è stato applicato in molti altri sistemi, inclusi-coltura a base di cellule sistemi 5,6 e altri sistemi modello in vivo 6-9. Qui si descrive l&#39;adattamento del metodo TAP in Drosophila. Per prima cosa generiamo pUAST-NTAP e pUAST-CTAP vettori per facilitare la clonazione e la fusione del tag TAP sia alla N-o C-terminale del gene di interesse. Il UAS-TAP-tag transgene viene poi espressa nel sistema nervoso sotto il controllo di un driver GAL4 neuronale 10. Successivamente, verrà raccolto un gran numero di teste mosca adulta, che hanno un alto contenuto di cellule neurali e sono facili da separare da altre parti del corpo dopo il congelamento sulla base delle differenze di dimensioni. Le teste adulti sono omogeneizzati e liquidati bcentrifugazioni sequenziali y, e il surnatante è soggetto a una procedura di TAP descritta di seguito.

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			<td height="42" style="height:42px;width:307px;">U.S.A. standard test sieve No. 25</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-18&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Kontes Dounce Tissue Grinders 15 ml</td>
			<td>Kimble Chase</td>
			<td>885300-0015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;"></td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG sepharose beads</td>
			<td>Pharmacia</td>
			<td style="width:119px;">17-0969-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.7 cm x 20 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4721</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.5 cm x 15 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4716</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calmodulin beads</td>
			<td style="width:104px;">Stratagene</td>
			<td style="width:119px;">214303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coors Mortar and Pestle</td>
			<td style="width:104px;">CoorsTek</td>
			<td>60311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AcTEV Protease</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:104px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Mix</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

Schermi genetici condotti utilizzando Drosophila melanogaster (moscerino della frutta) hanno fatto numerose scoperte pietra miliare nel progresso delle scienze biologiche. Tuttavia, l&#39;uso di schermi biochimiche finalizzate ad estendere le conoscenze acquisite da analisi genetica è stata esplorata solo di recente. Qui si descrive un metodo per purificare il complesso proteico che si associa con qualsiasi proteina di interesse da teste di mosca adulta. Questo metodo sfrutta il sistema Drosophila GAL4/UAS per esprimere una proteina esca fuso con un Tandem Affinity Purification (TAP) tag in neuroni di topo in vivo e quindi implementa due cicli di purificazione utilizzando una procedura TAP simile a quello inizialmente stabilito in lievito 1 per purificare il complesso proteina interagente. Al termine di questa procedura, una miscela di diversi complessi proteici si ottiene la cui identità molecolare può essere determinato mediante spettrometria di massa. Validazione delle proteine ​​candidati beneficeranno del reSource e facilità di esecuzione di perdita-di-funzione studi di mosche. Approcci simili possono essere applicati ad altri tessuti mosca. Crediamo che la combinazione di manipolazioni genetiche e questo approccio proteomico nel sistema modello fly detiene un enorme potenziale per affrontare i problemi fondamentali nel campo della neurobiologia e oltre.

Qui mostriamo il nostro sforzo per identificare le proteine ​​Highwire interagenti nel cervello della mosca. Highwire (HIW) e le sue vertebrati e invertebrati omologhi sono enormi ubiquitina ligasi che regolano lo sviluppo e la riparazione del sistema nervoso 14. Essi condividono un certo numero di domini funzionali altamente conservate. Tuttavia, le loro azioni molecolari non sono del tutto chiare. Lavoro fatto in verme, vola e mouse hanno portato a modello di lavoro corrente che funzioni hiw come ligasi...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP at JoVE.com

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila è famosa per la sua manipolazione genetica potente, ma non per la sua idoneità di analisi biochimica in profondità. Qui vi presentiamo una procedura basata TAP-per identificare interagiscono partner di qualsiasi proteina di interesse da parte del cervello della mosca. Questa procedura può potenzialmente portare a nuove vie di ricerca.

Identificare proteina-proteina interazione in<em&gt; Drosophila</em&gt; Adult capi di Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

identifying-protein-protein-interaction-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

13.8K Views.  Louisiana State University Health Sciences Center.  Drosophila è famosa per la sua manipolazione genetica potente, ma non per la sua idoneità di analisi biochimica in profondità. Qui vi presentiamo una procedura basata TAP-per identificare interagiscono partner di qualsiasi proteina di interesse da parte del cervello della mosca. Questa procedura può potenzialmente portare a nuove vie di ricerca. 

Video: Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificazione delle proteine ​​ortogonali Facilitato da una piastrina Affinity Bispecific

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Ricerca

Biologia

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Individuazione di partner proteine ​​interagenti mediante Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento di cromatina Purificazione RNA (disturbo)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificazione di complessi proteici in<em&gt; Escherichia coli</em&gt; Con sequenziale purificazione per affinità Peptide in combinazione con Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purificazione di Trascrizioni e metaboliti<em&gt; Drosophila</em&gt; Teste

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Protein Purification-libera Metodo di Binding Affinity Determinazione Thermophoresis microscala

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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

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

Identificare proteina-proteina siti di interazione Uso Peptide Array

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genome-wide proteina-proteina interazione Screening per Protein-frammento Complementation Assay (PCA) in cellule viventi

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...