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 ...

Overall goal of this procedure is to identify proteins interacting with a Nuclear Cofactor using LC-MS/MS Analysis. This method can help answer key questions related to transcriptional mechanisms in the gene regulation field. The main advantage of this technique is that we can identify the proteins interacting with a nuclear cofactor.Moreover, this method allows us to better understand the transcriptional regulatory mechanisms of the targeted nuclear factors. Harvest HEK 293 cells expressing flag-tagged ARIP4 36 hours after transvection by washing the cell sheet with ice cold PBS and collecting the cells with a cell scrapper. Transfer the cells into a 1.5 milliliter tube and then centrifuge at 8000g for two minutes at four degrees Celsius.Aspirate the supernatant and re-suspend the cell pellet in five times the pellet volume of High Salt Buffer. Rotate the mixture for 20 minutes at four degrees Celsius. After 20 minutes of rotation, centrifuge the tube at 17, 700g for 10 minutes at four degrees Celsius.Next, transfer the supernatants to a new two milliliter tube and add Dilution buffer to three times the volume of supernatant. Perform another cetrifugation and then transfer the supernatant to a new two milliliter tube. During the 10 minute centrifugation wash 50 microliters of anti-flag beads with PBS.Then centrifuge the tube at 2000g for one minute at four degrees Celsius. Remove the supernatant and repeat wash steps first with 1 molar glycine hydrochloride and then with one molar tris hydrochloride. Then wash twice with Low salt buffer.Next, mix 50 microliters of washed beads with whole-cell extracts. Gently rotate the mixture for four hours at two to four degrees Celsius. Then use a blunted tip to transfer the sample into a micro spin column and allow the solution to drip through into a 1.5 milliliter tube.Snap freeze the flow through in liquid nitrogen and store at minus 80 degrees Celsius. Next, wash the flag beads with Low salt buffer five times. To elute, place the column into a 1.5 milliliter tube and centrifuge at 2000g for one minute to dry the beads.Cap the bottom of the column and then add 50 microliters of 1 molar glycine hydrochloride. Gently shake the mixture for 10 minutes at room temperature. After 10 minutes, place the column into a new 1.5 milliliter tube and centrifuge at 2000g for one minute.Neutralize the eluted solution with 10 microliters of one molar tris hydrochloride and mix well by pipetting and vortexing. Add 50 microliters of 100 millimolar ammonium bicarbonate. 10 microliters of acetonitrile, two microliters of dithiothreitol and 50 microliters of neutralized sample.Incubate for 30 minutes at 56 degrees Celsius. After incubation, leave the sample at room temperature for 10 minutes. Then add 10 microliters of 333 millimolar iodoacetamide to the sample and incubate in the dark for 30 minutes at 37 degrees C.Add 10 microliters of trypsin to the mixture and incubate overnight at 37 degrees Celsius.The next day transport the final product to an LC-MS/MS facility or to a third party mass spectrometry lab for analysis. This image shows silver staining of flag epitope tagged ARIP4 expressed in HEK 293 cells after immunal precipitation with a flag specific antibody and separation by electrophoresis. As a control, a mock purification was performed on HEK 293 cells that did not express exogenous proteins.Using our method, it is possible to identify putative nuclear cofactors via their transcriptional factor of interest and further enhance our understanding of transcriptional regulatory mechanisms.

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