Name: a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis
Uploaded: 2017-01-24T12:30:00
Description: Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), the Takeda Science Foundation (HT), and by a Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) to HT.

1. Transfektion <ol><li> Seed HEK293-celler i 100 mm dyrkningsplader (2 x 10 6 celler / skål). Dyrke cellerne i Dulbeccos modificerede Eagles medium suppleret med 10% føtalt kalveserum, 100 ug / ml streptomycin, og 100 enheder / ml penicillin. Inkubér cellerne i en fugtig inkubator ved 5% CO2 og 37 ° C. </li><li> Den næste dag, transfektion cellerne med 10 ug FLAG-mærket ARIP4 plasmid hjælp 40 pi transfektionsreagens i overensstemmelse med producentens anvisninger 11.</su...

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Effektiv transfektion er afgørende for at opnå et vellykket resultat med denne protokol. Derfor anbefaler vi at bruge Western blot-analyse for at bestemme de immunopræcipiterede FLAG-mærkede protein niveauer. Dette trin giver brugerne mulighed for at kontrollere, at deres protein af interesse er korrekt overudtrykt og desuden, at IP blev udført med succes. FLAG-mærket protein niveauer bør også kontrolleres forud for massespektrometri analyse. En mock prøve bør anvendes som en negat...

Protein-protein interaktioner spiller en vigtig rolle i mange biologiske funktioner. Som sådan er disse interaktioner blevet impliceret i signaltransduktion; protein transport over cellemembraner; cellemetabolisme; og adskillige nukleare processer, herunder DNA-replikation, DNA beskadigelse reparation, rekombination og transkription 1, 2, 3, 4. Identifikation af proteiner involveret i disse interaktioner er derfor afgørende for at fremme vores forståelse af disse cellulære processer. Immunpræcipitering (IP) er en valideret teknik, der anvendes til at analysere protein-protein-interaktioner. For at lette identifikationen af co-immunpræcipiteret proteiner, er massespektrometri ofte brugt 5, 6, 7, 8,9. Ved at målrette en kendt medlem af et proteinkompleks med et antistof, er det muligt at isolere protein-kompleks og efterfølgende identificere sine ukendte komponenter via massespektrometrianalyse. ARIP4 (androgen receptor-interagerende protein 4), en transskriptionel coregulator, interagerer med nukleare receptorproteiner for at aktivere eller undertrykke sit mål initiativtagere i en kontekst-afhængig måde 9, 10. For bedre at forstå de transkriptionelle reguleringsmekanismer for disse nukleare faktorer, beskriver vi en omfattende metode til at rense og identificere ARIP4 interagerende proteiner ved hjælp af LC-MS / MS-system.

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			<td height="81" style="height:81px;width:216px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:71px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11668019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Protease Inhibitor Cocktail (EDTA free) (100x)</td>
			<td style="width:71px;">nacalai tesque</td>
			<td style="width:119px;">03969-21</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ANTI-FLAG M2 Affinity Gel</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Micro Bio-Spin Columns</td>
			<td style="width:71px;">BIO-RAD</td>
			<td style="width:119px;">732-6204</td>
			<td></td>
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a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

Vi identificerede en stærk ARIP4 signal, omkring 160 KDa, såvel som dem af en mock prøve kontrol og flere andre ukendte proteiner (figur 1). LC-MS / MS-analyse identificeret både ARIP4 komplekse peptider og de potentielle peptid cofaktorer inden den del af FLAG perler (tabel 1). P62 (Sequestosome1) blev en kendt ARIP4 cofaktor 11 identificeret i analysen (tabel 1), hvilket bekræfter effekten af dette system <...

Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Vi har etableret en fremgangsmåde til oprensning af coregulatoriske interaktion proteiner ved anvendelse af LC-MS / MS-system.

En Protein Forberedelse Metode til High-throughput Identifikation af proteiner Samspil med en Nuclear Cofactor Brug LC-MS / MS-analyse

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

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