Name: modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin
Uploaded: 2012-01-17T13:00:00
Description: Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Questo lavoro è stato finanziato dal NIH assegni di ricerca K01AR053210, R01AR061484 e una borsa di National Psoriasis Foundation.

1. Informazioni di base Il sistema del doppio ibrido di lievito è una potente tecnica genetica utilizzata per scoprire interazioni proteina-proteina 16, 17. Diversi tipi di 2-sistemi ibridi, come i sistemi basati su lexa, il sistema di reclutamento Sos, e batteri o cellule di mammifero a base di 2-ibridi, sono commerciali disponibili, questo articolo si concentra in particolare sulle modifiche dei più comunemente usati GAL4 base lievito 2-sistema ibrido. In breve, il metodo si bas...

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Doppio ibrido di lievito di screening ha dimostrato di essere uno strumento efficace per identificare interazioni proteina 16, 17. Rispetto ad altri approcci per identificare le proteine ​​di legame partner, come la biochimica di co-purificazione e proteine ​​chip, lievito sistema del doppio ibrido è un approccio sensibile genetico che può essere utilizzato per lo screening numero molto elevato di sequenze codificanti in un esperimento relativamente semplice, in Inoltre, rileva l&#39;interazione in ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> ProQuest sistema del doppio ibrido </td><td> Invitrogen </td><td> 10835 </td></tr><tr><td> YPD crescita media </td><td> Clontech </td><td> 630409 </td></tr><tr><td> YPD AGAR </td><td> Clontech </td><td> 630410 </td></tr><tr><td> Minimo SD agar Base </td><td> Clontech </td><td> 630412 </td></tr><tr><td> Leu-DO Supplemento </td><td> Clontech </td><td> 630414 </td></tr><tr><td> -Leu/-Trp DO Supplemento </td><td> Clontech </td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura DO Supplemento </td><td> Clontech </td><td> 630425 </td></tr><tr><td> 3-Amino-1 ,2,4-triazolo (3AT) </td><td> Sigma-Aldrich </td><td> A8056 </td></tr><tr><td> Luria brodo (LB) </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> X-Gal </td><td> Invitrogen </td><td> 15520-034 </td></tr><tr><td> Sonicato Salmon Sperm DNA </td><td> Stratagene </td><td> 201190 </td></tr><tr><td> Ampicillina </td><td> AMRESCO </td><td> 0339 </td></tr><tr><td> Kanamicina solfato </td><td> Invitrogen </td><td> 11815-024 </td></tr><tr><td> Efficienza subcloning ™ DH5α ™ cellule competenti </td><td> Invitrogen </td><td> 18265-017 </td></tr><tr><td> Trizma ® di base </td><td> Sigma-Aldrich </td><td> T6066 </td></tr><tr><td> Acetato di litio </td><td> Sigma-Aldrich </td><td> L4158 </td></tr><tr><td> Etilendiammintetraacetato </td><td> Sigma-Aldrich </td><td> ED </td></tr><tr><td> Membrana di nitrocellulosa </td><td> Bio-Rad </td><td> 162-0115 </td></tr><tr><td> 10-cm scatola di Petri </td><td> ITI scientifico </td><td> CT-903 </td></tr><tr><td> Incubatore (30 ° C) </td><td> ATR (Ecotron) </td><td></td></tr></table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulina (PGRN), noto anche come precursore granulin epithelin (GEP), è un fattore 593-amino-acido crescita autocrino. PGRN è nota per svolgere un ruolo critico in una varietà di processi fisiologici e malattie, tra cui l&#39;embriogenesi precoce, la guarigione della ferita 1, infiammazioni 2, 3, e ospitare difesa 4. PGRN funziona anche come un fattore neurotrofico 5, e le mutazioni nel gene PGRN con conseguente perdita parziale della proteina causano demenza frontotemporale PGRN 6, 7. I nostri studi recenti hanno portato all&#39;isolamento di PGRN come un importante regolatore di sviluppo della cartilagine e del degrado 8-11. Anche se PGRN, scoperto quasi due decenni fa, gioca un ruolo cruciale in molteplici condizioni fisiologiche e patologiche, gli sforzi per sfruttare le azioni di PGRN e comprendere i meccanismi coinvolti sono stati significativamente ostacolata dalla nostra incapacità di identificare il suo legame al recettore (s). Per risolvere questo problema, abbiamo sviluppato un modicato doppio ibrido di lievito (MY2H) approccio basato sulla più comunemente utilizzati GAL4 base 2-sistema ibrido. Rispetto al tradizionale doppio ibrido di lievito schermo, MY2H riduce drasticamente il processo di schermo e riduce il numero di cloni falsi positivi. Inoltre, questo approccio è riproducibile e affidabile, e abbiamo impiegato con successo questo sistema per isolare le proteine ​​leganti di esche, tra cui ioni di canale 12, proteine ​​della matrice extracellulare 10, 13, e del fattore di crescita 14. In questo lavoro, si descrive questa procedura MY2H sperimentale in dettaglio utilizzando PGRN come un esempio che ha portato alla identificazione di TNFR2 come la prima nota PGRN associata recettore 14, 15.

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Abbiamo modificato il lievito tradizionale doppio ibrido di screening, un efficace strumento genetico per identificare interazioni proteiche. Questa modifica riduce notevolmente il processo, riduce il carico di lavoro e, soprattutto, riduce il numero di falsi positivi. Inoltre, questo approccio è riproducibile ed affidabile.

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

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