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

Dette arbejde er finansieret af NIH forskningsbevillinger K01AR053210, R01AR061484 og et tilskud fra National Psoriasis Foundation.

1. Baggrundsinformation Den gær to-hybrid-system er en stærk genetisk teknik, der bruges til at opdage protein-protein interaktioner 16, 17. Flere typer af 2-hybrid systemer, såsom Lexa-baserede systemer, SOS Rekruttering System, og bakterier-eller pattedyr celle-baseret 2-hybrider, der er kommercielt tilgængelige, dette dokument specifikt fokuseres på ændringer af de mest almindeligt anvendte GAL4 baseret gær 2-hybrid-system. Kort fortalt, er metoden baseret på egenskaberne...

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Gær to-hybrid screening har vist sig at være et effektivt redskab til at identificere protein interaktioner 16, 17. Sammenlignet med andre metoder til at identificere proteinbindende partnere, såsom biokemiske co-rensning og protein chips, gær to-hybrid systemet er et følsomt genetisk tilgang, der kan bruges til screening meget stort antal kodningssekvenser i et relativt simpelt eksperiment, og i Desuden er det detekterer in vivo interaktion og behøver ikke kompliceret proteinoprensning. Selvfølgelig g...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> ProQuest to-hybrid-system </td><td> Invitrogen </td><td> 10835 </td></tr><tr><td> YPD vækstmedium </td><td> Clontech </td><td> 630409 </td></tr><tr><td> YPD Agar </td><td> Clontech </td><td> 630410 </td></tr><tr><td> Minimal SD agar Base </td><td> Clontech </td><td> 630412 </td></tr><tr><td> -Leu DO Supplement </td><td> Clontech </td><td> 630414 </td></tr><tr><td> -Leu/-Trp DO Supplement </td><td> Clontech </td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura DO Supplement </td><td> Clontech </td><td> 630425 </td></tr><tr><td> 3-Amino-1 ,2,4-triazol (3AT) </td><td> Sigma-Aldrich </td><td> A8056 </td></tr><tr><td> Luria-bouillon (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> Sonicated Laks Sperm DNA </td><td> Stratagene </td><td> 201190 </td></tr><tr><td> Ampicillin </td><td> AMRESCO </td><td> 0339 </td></tr><tr><td> Kanamycin Sulfate </td><td> Invitrogen </td><td> 11815-024 </td></tr><tr><td> Subcloning Effektivitet ™ DH5α ™ kompetente celler </td><td> Invitrogen </td><td> 18265-017 </td></tr><tr><td> Trizma ® basen </td><td> Sigma-Aldrich </td><td> T6066 </td></tr><tr><td> Lithium acetat </td><td> Sigma-Aldrich </td><td> L4158 </td></tr><tr><td> Ethylendiamintetra syre </td><td> Sigma-Aldrich </td><td> ED </td></tr><tr><td> Nitrocellulose Membran </td><td> Bio-Rad </td><td> 162-0115 </td></tr><tr><td> 10-cm petriskål </td><td> ITI Videnskabelige </td><td> CT-903 </td></tr><tr><td> Inkubator (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

Progranulin (PGRN), også kendt som granulin epithelin forløber (GEP), er en 593-amino-syre autocrine vækstfaktor. PGRN er kendt for at spille en afgørende rolle i en række forskellige fysiologisk og sygdom processer, herunder tidlige embryogenese, sårheling 1, inflammation 2, 3, og vært forsvar 4. PGRN fungerer også som en neurotrope faktor 5, og mutationer i PGRN genet resulterer i en delvis tab af PGRN protein årsag frontotemporal demens 6, 7. Vores seneste undersøgelser har ført til isolation af PGRN som en vigtig regulator af brusk udvikling og nedbrydning 8-11. Selvom PGRN, opdagede næsten to årtier siden, spiller afgørende roller i flere fysiologiske og patologiske tilstande, indsatsen for at udnytte handlinger PGRN og forstå de involverede mekanismer er blevet hæmmet af vores manglende evne til at identificere dens bindende receptor (r). For at løse dette problem, har vi udviklet en modificerede gær to-hybrid (MY2H) tilgang baseret på de mest almindeligt anvendte GAL4 baseret 2-hybrid-system. Sammenlignet med de konventionelle gær to-hybrid-skærm, dramatisk MY2H forkorter skærmen processen og reducerer antallet af falsk positive kloner. Hertil kommer, at denne tilgang reproducerbar og pålidelig, og vi har med succes anvendt dette system i at isolere proteiner af forskellige lokkemad, herunder ion-kanal 12, ekstracellulære matrix protein 10, 13, og vækstfaktor 14. I dette papir, beskriver vi denne MY2H eksperimentelle procedure i detaljer ved hjælp af PGRN som et eksempel, der førte til identifikationen af TNFR2 som den første kendte PGRN-associerede receptor 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

Vi har ændret det konventionelle gær to-hybrid screening, et effektivt genetisk redskab til at identificere protein interaktion. Denne ændring markant forkorter processen, reducerer arbejdsbyrden, og vigtigst, reducerer antallet af falske positiver. Hertil kommer, at denne tilgang reproducerbar og pålidelig.

Modificeret Gær-Two-Hybrid system til at identificere Proteiner Interaktion med Growth Factor Progranulin

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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