Name: proteomics to identify proteins interacting with p2x2 ligand-gated cation channels
Uploaded: 2009-05-18T23:00:00
Description: Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

SW和烟草花叶病毒是在美国国立卫生研究院支持的NCRR和NHLBI。 BSK和HS是由美国国立卫生研究院的NINDS和NIGMS支持。

实验步骤实验步骤（图2）包括四个部分，下面是一个分步进行的方式描述。 第1部分：亚克隆和表达P2X2受体的C -末端。 我们已经表达P2X2受体在细菌的C -末端全长识别脑蛋白与之结合。 <ol><li> C -末端（残基353-472）的P2X2受体（图1）经PCR扩增，克隆到PGEX 4NT1（GE生命科学）和测序验证。 </li><li>重组质粒转化E.表达重组蛋白的大...

<ol>
	<li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion channels of excitable membranes. , (2001).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors+and+ATP+signalling+at+synapses.">Molecular physiology of P2X receptors and ATP signalling at synapses.</a> Nat Rev Neurosci. 2, 165-174 (2001).</li><li>Egan, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+calcium+ions+to+P2X+channel+responses.">Contribution of calcium ions to P2X channel responses.</a> J Neurosci. 24, 3413-3420 (2004).</li><li>Burnashev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+permeability+of+ligand-gated+channels.">Calcium permeability of ligand-gated channels.</a> Cell Calcium. 24, 325-332 (1998).</li><li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131, 1047-1058 (2007).</li><li>Levitan, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+protein+complexes+associated+with+neuronal+ion+channels.">Signaling protein complexes associated with neuronal ion channels.</a> Nat Neurosci. 9, 305-310 (2006).</li><li>Khakh, B. S., North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X+receptors+as+cell-surface+ATP+sensors+in+health+and+disease.">P2X receptors as cell-surface ATP sensors in health and disease.</a> Nature. 442, 527-532 (2006).</li><li>Roger, S., Pelegrin, P., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+P2X7+receptor+currents+and+membrane+blebbing+via+constitutive+and+dynamic+calmodulin+binding.">Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding.</a> J Neurosci. 28, 6393-6401 (2008).</li><li>North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors.">Molecular physiology of P2X receptors.</a> Physiol Rev. 82, 1013-1067 (2002).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+union+of+pharmacology.+XXIV.+Current+status+of+the+nomenclature+and+properties+of+P2X+receptors+and+their+subunits..">International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits..</a> Pharmacol Rev. 53, 107-118 (2001).</li><li>Young, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+shape,+architecture+and+size+of+P2X4+receptors+determined+using+fluorescence+resonance+energy+transfer+and+electron+microscopy.">Molecular shape, architecture and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.</a> J Biol Chem. 283, 26241-26251 (2008).</li><li>Edmondson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+signaling+complexes+include+metabolism-+and+transcription/translation-related+proteins:+complimentary+separation+techniques+with+LC/MS/MS.">Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS.</a> Mol Cell Proteomics. 1, 421-433 (2002).</li><li>Evans, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthosteric+and+allosteric+binding+sites+of+P2X+receptors.">Orthosteric and allosteric binding sites of P2X receptors.</a> Eur Biophys J. 38, 319-327 (2009).</li></ol>

离子通道是一个不可分割的膜蛋白的主要类。它们包含装满水的毛孔，选择性地允许跨质膜离子的运动了他们的电化学梯度。离子通道门打开和关闭状态之间。 P2X配体门控离子通道的情况下发射器（如ATP），或触发门控步骤是，它可能是由与其他蛋白的相互作用调节。在过去的十年见证在我们了解如何P2X受体结合ATP的13增加，但仍了解较少的辅助蛋白，在调节P2X功能方面的作用。在本议?...

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<tr>
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<td>Reagent</td>
<td>JT Baker</td>
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<td>Reagent</td>
<td>BIO-RAD</td>
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<td>VW1507-01</td>
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<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
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<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
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<td>Adenosine Triphosphate (ATP)</td>
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<td>A7699</td>
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<td>Bradford reagent</td>
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<td>BIO-RAD</td>
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<td>Reagent</td>
<td>Fisher Scientific</td>
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<td>62971</td>
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<td>S7653</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
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<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
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<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
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<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
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<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
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<td>Sigma</td>
<td>T9281</td>
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<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
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<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
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<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
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<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
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<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
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<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
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proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

配体门控离子通道1在中枢神经系统的突触联系的基础。在哺乳动物中，有三个家庭的配体门控通道：循环的半胱氨酸，谷氨酸门控和P2X受体通道 2 。在每一种情况下变送器具有约束力，导致毛孔开放，通过离子流及其电化学梯度。许多配体门控通道，也渗透到钙离子的3，4，有下游信号的角色（如基因调控），可能会超过通道的开放时间。因此，配体门控通道信号在广泛的时间尺度从几毫秒到数天不等。鉴于这些重要的角色，有必要了解如何配体门控离子通道本身是由蛋白质所规管，以及如何将这些蛋白质可能调整的信号。最近的研究表明许多，如果不是全部的渠道可能是部分蛋白信号复合物6。在这篇文章中，我们将解释如何识别的蛋白质结合P2X2受体胞浆域的C -末端的各个方面。 P2X受体是ATP门控的阳离子通道，由7个亚基（P2X1，P2X7）组成。 P2X受体广泛表达在大脑中，在那里他们介导兴奋性突触传递和神经递质的释放 7的突触前便利。 P2X受体在兴奋和非兴奋性细胞介导的神经信号，炎症和心血管功能 8的关键角色。 P2X2受体在神经系统的9的丰富，是本研究的重点。每个P2X亚基被认为是拥有两个膜跨越段（TM1和TM2）的，由胞外区 7和细胞内的N和C末端（图1A）7分开。 P2X亚基10（P2X1，P2X7）显示30-50％的序列同源性氨基酸水平的 11 。 P2X受体只包含三个亚基，这是最简单的离子型受体的化学计量。 P2X2 C -末端由120个氨基酸（图1b）组成，并含有几种蛋白质对接的共识网站，支持这一假设P2X2受体信号复合物的一部分。不过，虽然有几个功能已被归因于P2X2受体9 C -末端没有研究描述分子的合作伙伴，这种蛋白质通过细胞内C -末端全长夫妇。在此方法纸中，我们描述了蛋白质组学的方法来识别与P2X2受体的C -末端全长相互作用的蛋白质。

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

我们描述一个简单的协议，以确定大脑的蛋白质结合ATP门控P2X2受体的C末端全长。这种方法的延伸和系统应用到所有P2X受体预计将导致一个更好地了解P2X受体信号。

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Research

JoVE Journal

Biology

Identification of protein complexes with quantitative proteomics in S. cerevisiae

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

科研

生物学

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

使用范围，以确定潜在的监管图案协同调控基因

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

自下而上和猎枪蛋白质组学鉴定一个全面的蛋白质组耳蜗

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

糖肽捕获的细胞表面蛋白组学

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...