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. अभिकर्मक <ol><li> 100 मिमी संस्कृति प्लेट (2 एक्स 10 6 कोशिकाओं / पकवान) में HEK293 कोशिकाओं बीज। संस्कृति Dulbecco संशोधित ईगल मध्यम में कोशिकाओं 10% भ्रूण बछड़ा सीरम, 100 माइक्रोग्राम / एमएल स्ट्रेप्टोमाइसिन, और 100 इकाइ?...

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कुशल अभिकर्मक इस प्रोटोकॉल के साथ एक सफल परिणाम प्राप्त करने के लिए आवश्यक है। तदनुसार, हम पश्चिमी धब्बा विश्लेषण का उपयोग immunoprecipitated झंडा टैग प्रोटीन का स्तर निर्धारित करने के लिए सलाह देते हैं। यह कदम उ?...

प्रोटीन, प्रोटीन बातचीत में कई जैविक कार्यों में एक महत्वपूर्ण भूमिका निभाते हैं। जैसे, इन मुलाकातों संकेत पारगमन में फंसाया गया है; कोशिका झिल्ली भर में प्रोटीन परिवहन; सेल चयापचय; और डीएनए प्रतिकृति, डीएनए की क्षति की मरम्मत, पुनर्संयोजन, और प्रतिलेखन 1, 2, 3, 4 सहित कई परमाणु प्रक्रियाओं। इन मुलाकातों में शामिल प्रोटीन की पहचान करना इसलिए इन सेलुलर प्रक्रियाओं के बारे में हमारी समझ को आगे बढ़ाने के लिए महत्वपूर्ण है। Immunoprecipitation (आईपी) प्रोटीन, प्रोटीन बातचीत का विश्लेषण करने के लिए इस्तेमाल एक मान्य तकनीक है। आदेश सह immunoprecipitated प्रोटीन की पहचान की सुविधा के लिए, मास स्पेक्ट्रोमेट्री अक्सर उपयोग किया जाता है 5, 6, 7, 8,9। एक एंटीबॉडी के साथ एक प्रोटीन परिसर के एक ज्ञात सदस्य को लक्षित करके, यह प्रोटीन जटिल अलग-थलग करने और बाद में मास स्पेक्ट्रोमेट्री विश्लेषण के माध्यम से अपनी अज्ञात घटकों की पहचान करने के लिए संभव है। ARIP4 (एण्ड्रोजन रिसेप्टर बातचीत प्रोटीन 4), एक ट्रांसक्रिप्शनल coregulator, आदेश में सक्रिय या एक संदर्भ पर निर्भर ढंग से 9, 10 में अपने लक्ष्य के प्रमोटरों को दबाने के लिए परमाणु रिसेप्टर प्रोटीन के साथ सूचना का आदान प्रदान। बेहतर इन परमाणु कारकों गवर्निंग विनियामक तंत्र को समझने के लिए, हम शुद्ध और नियंत्रण रेखा एमएस / एमएस प्रणाली का उपयोग करके ARIP4 बातचीत प्रोटीन की पहचान करने के लिए एक व्यापक विधि का वर्णन है।

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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.

हम एक मजबूत संकेत ARIP4, लगभग 160 केडीए, के रूप में अच्छी तरह से एक नकली नमूना नियंत्रण और कई अन्य अज्ञात प्रोटीन के रूप में उन (चित्रा 1) की पहचान की। नियंत्रण रेखा एमएस / एमएस विश्लेषण दोनों AR...

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

हम नियंत्रण रेखा एमएस / एमएस प्रणाली का उपयोग कर coregulatory बातचीत प्रोटीन की शुद्धि के लिए एक विधि की स्थापना की है।

एक प्रोटीन प्रोटीन की उच्च throughput पहचान नियंत्रण रेखा एमएस / एमएस विश्लेषण का प्रयोग एक परमाणु cofactor साथ बातचीत के लिए तैयारी विधि

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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उच्च प्रवाह, मात्रात्मक डॉट दाग विश्लेषण का उपयोग ऊतक के स्तर पर एक चयनित प्रोटीन की सामग्री का निरपेक्ष निर्धारण (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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