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. A transfecção <ol><li> Semente células HEK293 em placas de cultura de 100 mm (2 x 10 6 células / prato). Cultura das células em meio de Eagle modificado por Dulbecco suplementado com 10% de soro fetal de vitelo, 100 ug / ml de estreptomicina, e 100 unidades / ml de penicilina. Incubam-se as células numa incubadora humidificada a 5% CO 2 e 37 ° C. </li><li> No dia seguinte, transfectar as células com 10 ug de plasmídeo ARIP4 marcado com FLAG, utilizando 40 ul de reagente de transfecç?...

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transfecção eficiente é essencial para obter um resultado bem sucedido com este protocolo. Assim, recomendamos o uso de análise de Western blot para determinar os níveis de proteína FLAG-marcadas imunoprecipitadas. Esta etapa permite que os usuários para verificar se a sua proteína de interesse é adequadamente overexpressed e, além disso, que o IP foi realizada com sucesso. Os níveis de proteína marcada com Flag também deve ser verificado antes da análise por espectrometria de massa. <p class="jove_con...

interacções proteína-proteína desempenham um papel importante em muitas funções biológicas. Como tal, estas interacções têm sido implicados na transdução de sinal; transporte de proteínas através das membranas celulares; metabolismo celular; e vários processos nucleares, incluindo a replicação do ADN, reparação de danos do ADN, a recombinação e a transcrição 1, 2, 3, 4. Identificar as proteínas envolvidas nestas interacções é, portanto, fundamental para o avanço da nossa compreensão destes processos celulares. A imunoprecipitação (IP) é uma técnica validado e utilizado para analisar interacções proteína-proteína. A fim de facilitar a identificação de proteínas co-imunoprecipitada, a espectrometria de massa é frequentemente utilizada 5, 6, 7, 8,9. Ao direccionar um membro conhecido de um complexo de proteína com um anticorpo, é possível isolar o complexo de proteína e, subsequentemente, para identificar os seus componentes desconhecidos através de análise de espectrometria de massa. ARIP4 (proteína-receptor de androgénio interagindo 4), um coregulator transcricional, interage com as proteínas nucleares do receptor, a fim de activar ou reprimir seus promotores-alvo de uma forma dependente do contexto 9, 10. Para melhor compreender os mecanismos reguladores da transcrição que regulam estes factores nucleares, nós descrevemos um método global para purificar e identificar proteínas que interagem ARIP4 usando o sistema de LC-MS / MS.

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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>
		</tr>
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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.

Foram identificados um sinal forte ARIP4, cerca de 160 kDa, bem como aqueles de uma amostra de controlo simulado e várias outras proteínas desconhecidas (Figura 1). A análise por LC-MS / MS identificou ambos os péptidos complexos ARIP4 e os potenciais co-factores peptídicos dentro da fracção de grânulos de bandeira (Tabela 1). p62 (Sequestosome1), um cofactor ARIP4 conhecido foi 11 identificada na análise (Tabela ...

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

Nós estabelecemos um método para a purificação de proteínas de interacção co-regulação que utilizam o sistema de LC-MS / MS.

Uma proteína Método de Preparação para a Identificação de alto rendimento de proteínas que interagem com um co-fator nuclear utilizando LC-MS / MS Análise

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.

Research

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