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. La transfección <ol><li> Sembrar las células HEK293 en placas de cultivo de 100 mm (2 x 10 6 células / placa). Cultivar las células en medio de Eagle modificado por Dulbecco suplementado con 10% de suero de ternera fetal, 100 mg / ml de estreptomicina y 100 unidades / ml de penicilina. Se incuban las células en un incubador humidificado a 5% de CO2 y 37 ° C. </li><li> El día siguiente, transfectar las células con 10 g de plásmido ARIP4 marcado con FLAG utilizando 40 l de reactivo de t...

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transfección eficiente es esencial para obtener un resultado exitoso con este protocolo. En consecuencia, se recomienda el uso de Western blot para determinar los niveles de proteína marcada con FLAG inmunoprecipitadas. Este paso permite a los usuarios para verificar que su proteína de interés se sobreexpresa adecuadamente y, por otra parte, que la IP se realizó con éxito. los niveles de proteína marcada con FLAG también deben ser revisadas antes del análisis de espectrometría de masas. <p class="jove_cont...

Las interacciones proteína-proteína desempeñan un papel importante en muchas funciones biológicas. Como tal, estas interacciones se han implicado en la transducción de señales; el transporte de proteínas a través de las membranas celulares; el metabolismo celular; y varios procesos nucleares, incluyendo la replicación del ADN, el ADN a reparar el daño, la recombinación y la transcripción 1, 2, 3, 4. La identificación de las proteínas implicadas en estas interacciones tanto, es fundamental para avanzar en nuestra comprensión de estos procesos celulares. Inmunoprecipitación (IP) es una técnica validado utilizado para analizar interacciones proteína-proteína. Con el fin de facilitar la identificación de las proteínas co-inmunoprecipitado, espectrometría de masas se utiliza a menudo 5, 6, 7, 8,9. Al dirigirse a un miembro conocido de un complejo de proteínas con un anticuerpo, es posible aislar el complejo de la proteína y para identificar posteriormente sus componentes desconocidos a través de análisis de espectrometría de masas. ARIP4 (andrógeno proteína del receptor de interacción 4), una coregulator transcripcional, interactúa con las proteínas del receptor nuclear con el fin de activar o reprimir sus promotores diana de una manera dependiente del contexto 9, 10. Para comprender mejor los mecanismos reguladores de la transcripción que regulan estos factores nucleares, se describe un método integral para purificar e identificar las proteínas que interactúan ARIP4 utilizando el sistema 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>
	</tbody>
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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.

Se identificaron una fuerte señal ARIP4, alrededor de 160 KDa, así como las de un control de ejemplo mock y varias otras proteínas desconocidas (Figura 1). Análisis de LC-MS / MS identificó tanto los péptidos complejos ARIP4 y los cofactores de péptidos potenciales dentro de la fracción de perlas de FLAG (Tabla 1). p62 (Sequestosome1), un conocido cofactor ARIP4 11 fue identificado en el análisis (Tabla 1),</stron...

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

Hemos establecido un método para la purificación de proteínas de interacción correguladoras utilizando el sistema de LC-MS / MS.

Una proteína Preparación método para la identificación de alto rendimiento de proteínas que interactúan con un cofactor nuclear utilizando LC-MS / MS análisis

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

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