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. 형질 <ol><li> 100 mm 배양 판 (2 × 106 세포 / 접시)에 HEK293 세포 종자. 배양 10 % 소 태아 혈청, 100 μg의 / ㎖ 스트렙토 마이신, 100 유닛 / ㎖의 페니실린으로 보충 된 둘 베코 변형 이글 배지에서 세포. 5 % CO 2 및 37 ℃로 가습 된 배양기에서 세포를 인큐베이션. </li><li> 다음 날, FLAG-태그 ARIP4 플라스미드의 10 μg의 제조업체의 방향 (11)에 따라 형질 전환 시약의 40 μl를 ?...

<ol>
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효율적인 형질 감염 프로토콜이 성공적인 결과를 얻는 것이 필수적이다. 따라서, 우리는 면역 FLAG 태그가 단백질 수준을 결정하는 웨스턴 블롯 분석을 사용하는 것이 좋습니다. 이 단계는 IP가 성공적으로 수행되었는지, 그들의 관심 단백질이 제대로 또한, 과발현되어 있는지 확인하려면 사용자 수 있습니다. FLAG 태그가 단백질 수준은 질량 분광 분석에 앞서 확인하여야한다. ...

단백질 - 단백질 상호 작용은 다양한 생물학적 기능에 중요한 역할을한다. 따라서, 이러한 상호 작용은 신호 전달에 연루되어왔다 세포막 단백질 수송; 세포의 신진 대사; 및 DNA 복제, DNA 손상 복구, 재조합 및 전사 1, 2, 3, 4 등 여러 핵 프로세스. 이러한 상호 작용에 관여하는 단백질을 확인하는 것은이 세포 과정에 대한 우리의 이해를 증진하는 것이 중요하다. 면역 침전 (IP)는 단백질 - 단백질 상호 작용을 분석하는데 사용되는 검증 된 기술이다. 공동 - 면역 침전 된 단백질의 동정을 용이하게하기 위하여, 질량 분석계는 종종 이용 5, 6, 7, 8,9. 항체와 단백질 복합체의 공지 부재 타겟팅함으로써, 단백질 복합체를 분리하고,이어서 질량 분광 분석을 통해 그 미지의 컴포넌트를 식별 할 수있다. ARIP4 (안드로겐 수용체 상호 작용하는 단백질 4), 전사 coregulator은 활성화 또는 상황에 의존적으로 9, 10 년 대상 발기인을 억제하기 위해 핵 수용체 단백질과 상호 작용한다. 더 이러한 핵 인자를 지배하는 전사 조절 메커니즘을 이해하기 위해 정화하고, LC-MS / MS 시스템을 사용하여 ARIP4 상호 작용 단백질을 확인하는 포괄적 인 방법을 설명한다.

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		<tr height="81">
			<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.

우리는 강한 신호 ARIP4 약 160 KDa의뿐 모의 샘플 제어 및 몇몇 다른 미지의 단백질의 것과 (도 1)을 확인 하였다. LC-MS / MS 분석은 펩티드 복합체 ARIP4 FLAG 비드 (표 1)의 분획 내의 잠재적 인 펩티드 공동 인자를 모두 식별. P62 (Sequestosome1)는 공지 ARIP4 공동 인자 (11)는이 시스템 (11)의 유효성을 확인하는 분석 (표...

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

우리는 LC-MS / MS 시스템을 사용 coregulatory 상호 작용 단백질의 정제 방법을 확립 하였다.

LC-MS / MS 분석을 이용하여 핵 공동 인자와 상호 작용 단백질의 높은 처리량 식별 단백질 제조 방법

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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방법 및 HCN1 TRIP8b 간의 단백질 - 단백질 상호 작용의 작은 분자 억제제 식별

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

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Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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높은 처리량, 양적 도트를 사용 하 여 조직 수준에서 선택 된 단백질의 내용의 절대 결정 오 점 분석 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

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Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

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