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. Transfectie <ol><li> Zaad HEK293 cellen in 100 mm kweekplaten (2 x 10 6 cellen / schaal). Cultuur van de cellen in Dulbecco&#39;s gemodificeerd Eagle&#39;s medium gesupplementeerd met 10% foetaal kalfsserum, 100 ug / ml streptomycine en 100 eenheden / ml penicilline. Incubeer de cellen in een vochtige incubator bij 5% CO2 en 37 ° C. </li><li> De volgende dag transfecteren de cellen met 10 ug-FLAG tag ARIP4 plasmide middels 40 gl transfectiereagens volgens de instructies van de fabrikant <sup ...

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Efficiënte transfectie is essentieel voor een succesvol resultaat met dit protocol verkregen. Daarom raden wij u aan Western blot analyse om de immunogeprecipiteerd FLAG-gemerkte eiwitten te bepalen. Deze stap kan de gebruiker controleren of hun eiwit van interesse behoren tot overexpressie wordt gebracht en bovendien dat de IP is gelukt. FLAG-gemerkt eiwit niveaus ook vóór de massaspectrometrie analyse gecontroleerd. Een mock monster moet worden gebruikt als een negatieve controle te ver...

Eiwit-eiwit interacties een belangrijke rol in vele biologische functies. Als zodanig zijn deze interacties betrokken bij signaaltransductie; eiwit transport over celmembranen; celstofwisseling; en verschillende nucleaire processen, waaronder DNA-replicatie, DNA schade herstel, recombinatie en transcriptie 1, 2, 3, 4. Het identificeren van de eiwitten die betrokken zijn bij deze interacties is daarom van cruciaal belang voor onze kennis van deze cellulaire processen. Immunoprecipitatie (IP) is een gevalideerde techniek om eiwit-eiwit interacties analyseren. Om de identificatie van co-immunogeprecipiteerd eiwitten vergemakkelijken, wordt vaak gebruikt massaspectrometrie 5, 6, 7, 8,9. Door zich te richten een bekend lid van een eiwitcomplex met een antilichaam, is het mogelijk om het eiwitcomplex te isoleren en vervolgens de onbekende componenten identificeren via massaspectrometrie analyse. ARIP4 (androgeen-receptor interactie eiwit 4), een transcriptie coregulator samenwerkt met nucleaire receptoreiwitten voor het activeren of onderdrukken zijn doel promotors in een context-afhankelijke wijze 9, 10. Om beter te begrijpen van de transcriptie regulerende mechanismen die deze nucleaire factoren beschrijven we een uitgebreide methode te zuiveren en te identificeren ARIP4 interagerende eiwitten met behulp van de LC-MS / MS-systeem.

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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.

We identificeerden een sterk signaal ARIP4, ongeveer 160 kDa, evenals die van een mock vergelijkingsmonster en verscheidene andere onbekende eiwitten (Figuur 1). LC-MS / MS analyse identificeerde zowel ARIP4 complex peptiden en de potentiële peptide cofactoren in de fractie van FLAG parels (tabel 1). p62 (Sequestosome1), een bekende ARIP4 cofactor 11 werd geïdentificeerd in de analyse (tabel 1), waarbij de werkz...

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

We hebben een werkwijze voor het zuiveren van eiwitten mederegulerende interactie met de LC-MS / MS systeem.

Een eiwit Bereidingswijze voor de High-throughput identificatie van eiwitten Interactie met een nucleair Cofactor Met behulp van LC-MS / MS Analysis

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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