Name: acyl-pegyl exchange gel shift assay for quantitative determination of palmitoylation of brain membrane proteins
Uploaded: 2020-03-29T14:00:00
Description: Watch this Scientific Journal Video about Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins at JoVE.com

作者感谢K.Woolfrey对APEGS测定的建议和意见。这些研究由白厅基金会和NIH-NIMH（1R01MH119347）对E.D.的研究资助资助。

所有动物程序都遵循国家卫生研究院 （NIH） 制定的指导方针，并经加州大学戴维斯分校机构动物护理和使用委员会批准。
1. 小鼠脑膜的制备
<ol><li>使用断头器将小鼠斩首，并迅速解剖大脑（如果可能，在+lt;1 分钟内，以尽量减少解剖过程中可能发生的棕榈岛化变化）。在冰上的玻璃均质器（+12冲程）中，在10 mL的均质缓冲液（表1）中立即均质化。</li>
	<li>在4°...

<ol>
	<li>Blaskovic, S., Blanc, M., van der Goot, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+S-palmitoylation+do+to+membrane+proteins?.">What does S-palmitoylation do to membrane proteins?.</a> FEBS Journal. 280, 2766-2774 (2013).</li><li>Fukata, Y., Fukata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+palmitoylation+in+neuronal+development+and+synaptic+plasticity.">Protein palmitoylation in neuronal development and synaptic plasticity.</a> Nature Reviews Neuroscience. 11, 161-175 (2010).</li><li>Globa, A. K., Bamji, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+palmitoylation+in+the+development+and+plasticity+of+neuronal+connections.">Protein palmitoylation in the development and plasticity of neuronal connections.</a> Current Opinion in Neurobiology. 45, 210-220 (2017).</li><li>Thomas, G. M., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylation-dependent+regulation+of+glutamate+receptors+and+their+PDZ+domain-containing+partners.">Palmitoylation-dependent regulation of glutamate receptors and their PDZ domain-containing partners.</a> Biochemical Society Transactions. 41, 72-78 (2013).</li><li>Kalashnikova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SynDIG1:+an+activity-regulated,+AMPA-+receptor-interacting+transmembrane+protein+that+regulates+excitatory+synapse+development.">SynDIG1: an activity-regulated, AMPA- receptor-interacting transmembrane protein that regulates excitatory synapse development.</a> Neuron. 65, 80-93 (2010).</li><li>Kaur, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-Dependent+Palmitoylation+Controls+SynDIG1+Stability,+Localization,+and+Function.">Activity-Dependent Palmitoylation Controls SynDIG1 Stability, Localization, and Function.</a> Journal of Neuroscience. 36, 7562-7568 (2016).</li><li>Wan, J., Roth, A. F., Bailey, A. O., Davis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylated+proteins:+purification+and+identification.">Palmitoylated proteins: purification and identification.</a> Nature Protocols. 2, 1573-1584 (2007).</li><li>Howie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+recognition+by+the+cell+surface+palmitoyl+transferase+DHHC5.">Substrate recognition by the cell surface palmitoyl transferase DHHC5.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 17534-17539 (2014).</li><li>Kanadome, T., Yokoi, N., Fukata, Y., Fukata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Screening+of+Depalmitoylating+Enzymes+and+Evaluation+of+Their+Activities+by+the+Acyl-PEGyl+Exchange+Gel-Shift+(APEGS)+Assay.">Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay.</a> Methods in Molecular Biology. 2009, 83-98 (2019).</li><li>Percher, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-tag+labeling+reveals+site-specific+and+endogenous+levels+of+protein+S-fatty+acylation.">Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 113, 4302-4307 (2016).</li><li>Percher, A., Thinon, E., Hang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-Tag+Labeling+Using+Acyl-PEG+Exchange+for+the+Determination+of+Endogenous+Protein+S-Fatty+Acylation.">Mass-Tag Labeling Using Acyl-PEG Exchange for the Determination of Endogenous Protein S-Fatty Acylation.</a> Current Protocols in Protein Science. 89, 14.17.1-14.17.11 (2017).</li><li>Yokoi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+PSD-95+Depalmitoylating+Enzymes.">Identification of PSD-95 Depalmitoylating Enzymes.</a> Journal of Neuroscience. 36, 6431-6444 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Separating+Protein+with+SDS-PAGE.">JoVE Science Education Database. Separating Protein with SDS-PAGE.</a> Basic Methods in Cellular and Molecular Biology. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science+Education+Database.+The+Western+Blot.">Science Education Database. The Western Blot.</a> Basic Methods in Cellular and Molecular Biology. , (2019).</li><li>Chenaux, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+SynDIG1+Reduces+Excitatory+Synapse+Maturation+But+Not+Formation+In+Vivo.">Loss of SynDIG1 Reduces Excitatory Synapse Maturation But Not Formation In Vivo.</a> eNeuro. 3 (5), (2016).</li><li>Matt, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SynDIG4/Prrt1+Is+Required+for+Excitatory+Synapse+Development+and+Plasticity+Underlying+Cognitive+Function.">SynDIG4/Prrt1 Is Required for Excitatory Synapse Development and Plasticity Underlying Cognitive Function.</a> Cell Reports. 22, 2246-2253 (2018).</li><li>Purkey, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AKAP150+Palmitoylation+Regulates+Synaptic+Incorporation+of+Ca(2+)-Permeable+AMPA+Receptors+to+Control+LTP.">AKAP150 Palmitoylation Regulates Synaptic Incorporation of Ca(2+)-Permeable AMPA Receptors to Control LTP.</a> Cell Reports. 25, 974-987 (2018).</li><li>Woolfrey, K. M., Sanderson, J. L., Dell'Acqua, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+palmitoyl+acyltransferase+DHHC2+regulates+recycling+endosome+exocytosis+and+synaptic+potentiation+through+palmitoylation+of+AKAP79/150.">The palmitoyl acyltransferase DHHC2 regulates recycling endosome exocytosis and synaptic potentiation through palmitoylation of AKAP79/150.</a> Journal of Neuroscience. 35, 442-456 (2015).</li></ol>

在之前的工作中，我们利用 ABE 测定来证明 SynDIG1 在两种保存的共聚膜细胞残渣（存在于所有 SynDIG 蛋白质中）中以活动相关的方式进行棕榈酸化，以调节稳定性、定位性和功能6。一个限制是ABE测定需要亲和纯化与阿甘丝树脂结合作为程序的最后一步，导致信号的重大丢失，使定量分析复杂化。此外，ABE测定无法区分蛋白质是棕榈酸酯一次还是在多个地点。
...

S-掌上酸化是一种可逆的转化后靶蛋白，调节稳定的膜关联、蛋白质贩运和蛋白质-蛋白质相互作用1。它涉及通过棕榈酸乙酰乙酰转移酶（PAT）酶催化的硫化物链接，将16碳棕榈酸酶添加到半胱氨酸残留物中。大脑中的许多突触蛋白被棕榈酸化，包括AMPA-R和PSD-95，以活动依赖的方式调节稳定性、定位性和功能22，3，4。3,4通过辅助因素与突触支架（如 PSD-95）之间的相互作用，PSD 中突触性抗量系数水平的变化，导致突触可塑性;因此，确定突触蛋白的棕榈化状态的方法为突触可塑性机制提供了重要的见解。
之前，我们确定了SynDIG家族的四个基因（SynDIG1-4），编码与AmpARs5相关的大脑特异性跨膜蛋白。在分离的大鼠河马神经元中，SynDIG1的过度表达或击倒增加或减少，AMPA-R突触的大小和数量与使用免疫细胞化学和电生理学5检测到的+50%。我们利用acyl-生物素交换（ABE）测定法来证明SynDIG1在两种保存的共聚体-转膜细胞残留物（存在于所有SynDIG蛋白中）中，以活动相关的方式调节稳定性、定位性和功能6。ABE测定依赖于通过修饰和随后的亲和力纯化7保护的半胱氨酸的生物组。在这里，我们描述了一种创新的生化方法，acyl-PEGyl交换凝胶移位（APEGS）测定88，9，10，11，12，它不需要亲和力纯化，而是利用凝胶流动性的变化来确定感兴趣的蛋白质的修改次数。,9,10,11,12该协议被描述为研究小鼠大脑中的内源膜蛋白，适合其抗体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydroxylamine (HAM)</td>
			<td>ThermoFisher</td>
			<td>26103</td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxy-PEG-(CH2)3NHCO(CH2)2-MAL (mPEG)</td>
			<td>NOF</td>
			<td>ME-050MA</td>
			<td>MW ~5000 kDa</td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>N-ethylmalemide (NEM)</td>
			<td>Calbiochem</td>
			<td>34115</td>
			<td>Highly toxic.</td>
		</tr>
		<tr>
			<td>Optical imager for densitometry</td>
			<td>Azure Biosystems</td>
			<td>Sapphire Biomolecular Imager</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Polypropylene tubes with cap</td>
			<td>Fisher Scientific</td>
			<td>14-956-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets (glass)</td>
			<td>Fisher Scientific</td>
			<td>13-678-27D</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Beckman</td>
			<td>Allegra X-15R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl) phosphine-hydrochloride (TCEP)</td>
			<td>EMD Millipore</td>
			<td>580560</td>
			<td></td>
		</tr>
	</tbody>
</table>

acyl-pegyl exchange gel shift assay for quantitative determination of palmitoylation of brain membrane proteins

在脑后密度 （PSD） 中突触性 AMPA 受体 （AMPA） 水平的活动相关变化被认为是一种细胞机制的学习和记忆。棕榈化以活动相关的方式调节许多突触蛋白的定位和功能，包括AMPA-R、辅助因子和突触支架。我们确定了突触分化诱导基因（SynDIG）的四个基因系列（SynDIG1-4），编码与AmpARs关联并调节突触强度的特定于大脑的跨膜蛋白。SynDIG1 在位于并列跨膜区域位置 191 和 192 处的两个半胱氨酸残留物处进行棕榈酸酯化，对活动依赖性兴奋突触发育非常重要。在这里，我们描述了一种创新的生化方法，即acyl-PEGyl交换凝胶移位（APEGS）测定，以调查任何感兴趣的蛋白质的棕榈酸化状态，并证明其与小鼠脑水化中蛋白质的SynDIG家族的作用。

与不包括HAM的样品相比，用抗体对感兴趣的蛋白质进行免疫膨胀，可以揭示小鼠脑解解液中的棕榈酸化状态（非、单独、双重等）。此前，我们已经证明SynDIG1是使用ABE测定6在两个地点进行棕榈酸盐测定的;然而，我们无法确定这两个位点是否在脑组织中被修改。在这里，我们显示，SynDIG1，SynDIG4/Prrt1，和Prrt2在小鼠大脑中被棕榈酸化，他们有两个潜在的棕...

Watch this Scientific Journal Video about Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins at JoVE.com

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

棕榈化需要以可逆的方式将16碳棕榈酰化物与靶蛋白的半胱氨酸残留物结合。在这里，我们描述了一种生化方法，即acyl-PEGyl交换凝胶移位（APEGS）测定，以研究小鼠脑水化剂中任何感兴趣的蛋白质的棕榈酸化状态。

阿西尔-PEGyl交换凝胶移位测定，用于脑膜蛋白棕榈酸化定量测定

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

这种生化方法允许确定大脑中任何有适当抗体的膜蛋白的棕榈基化状态。这种技术的主要优点是它不需要亲和力纯化，而是利用凝胶流动性的变化来确定感兴趣的蛋白质的修饰数量。解剖大脑后，立即在冰面玻璃均质器中的10毫升均质缓冲液中均质。使用大约 12 冲程，在 1400 倍 G 和 4 摄氏度下将酸盐离心 15 分钟。将上一液转移到冰上的新管子上。将颗粒重新在10毫升的均质缓冲液中。使用大约六个笔画进行同质化。将悬浮液在710倍G和4摄氏度下离心10分钟。将超级natant将它们与4万倍G和4摄氏度的离心器结合20分钟。丢弃上流剂，通过用移液器尖端分解颗粒并上下移液，在均质缓冲液中重新填充颗粒膜分数。执行 ABCA 测定以量化蛋白质水平。要执行 APEGS 测定，将 470 微升缓冲液 A 放在 1.5 毫升管中，并加入约 100 至 200 毫克蛋白质。在25摄氏度和至少13，000次G下离心管，将不溶性蛋白质分离，然后分离出不溶性蛋白质，10分钟。将溶解的蛋白质转移到新的1.5毫升管中。首先，在55摄氏度下，在25微升TCEP中孵育溶解蛋白60分钟，从而破坏二硫化物键。然后，在室温下用12.5微升的NEM库存溶液孵育溶液，阻断释放半胱氨酸，长达3小时。接下来，开始氯仿甲醇沉淀过程。将蛋白质溶液从1.5毫升管转移到聚丙烯或玻璃管中，这种管可以以适中的速度在摆动桶转子中离心。短暂添加两毫升甲醇和涡流。短暂加入一毫升氯仿和涡流。然后加入1.5毫升的去维化水和涡流再次短暂。如有必要，反转管子以确保彻底混合。在摆动桶转子中，在 3000 倍 G 和 25 摄氏度下将样品离心 30 分钟。小心地取出并丢弃管中溶液的上相。加入1.5毫升甲醇。轻轻但彻底混合通过轻轻反转的管，小心不要分裂不透明的蛋白质煎饼。在摆动桶转子中，以 3000 倍 G 和 25 摄氏度的离心样品 10 分钟。使用玻璃血清移液器，在不干扰蛋白质煎饼的情况下，尽可能去除溶液的顶相。用一毫升甲醇小心地冲洗颗粒，让其风干至少10分钟。随着氯仿甲醇沉淀的完成，开始棕榈基硫化酯连接的裂解，将沉淀的蛋白质重新沉淀在缓冲液 A 的125微升中。转移到新的1.5毫升管中。将管短暂化，然后在13，000倍G或更高、25摄氏度时离心10分钟，以去除不溶性物质。将溶解蛋白转移到新的1.5毫升管中，并加入375微升缓冲液H或缓冲液T.在室温下孵育样品60分钟后，重复氯仿甲醇沉淀。要将 m-PEG 添加到未受保护的半胱氨酸中，首先在含有 10 密耳或更多 TCEP 的 100 微升缓冲液 A 中重新填充颗粒。然后将悬浮液转移到1.5毫升管中。添加 25 微升库存 m-PEG 5K 溶液，通过移液混合。在室温下孵育，端过端旋转。孵育60分钟后，通过再次进行氯仿甲醇沉淀，去除未合并的m-PEG 5K。在超过13，000倍的G和25摄氏度的离心机10分钟。小心地去除上相，避免厚，絮凝的煎饼。加入一毫升甲醇，轻轻但彻底混合。在超过13，000倍的G和25摄氏度的离心机10分钟。小心地取出上清液，用一毫升甲醇冲洗颗粒。同样，离心机在大于13，000倍的G和25摄氏度10分钟。空气干燥颗粒后，在50微升缓冲液 A 中重新排放，无需 TCEP 或任何还原剂。保留5微升溶液，用于BBA蛋白定量。定量后，添加适当量的 4x Laemmli 样品缓冲液。将样品加载到 SDS 页面凝胶上。使用标准分离传输和检测协议进行西印。为了研究SynDIG蛋白质的手掌化状态，对来自同质小鼠大脑的P2膜分数进行了PEGS测定。分离和与抗体的探测表明，SynDIG1、SynDIG4Prrt1和Prrt2在小鼠大脑中被棕榈化。氯仿甲醇沉淀的目的是防止一种化学物质，如NEM，干扰协议的下一步。适当的氯仿甲醇沉淀将去除不良化学物质，同时最大限度地减少蛋白质损失。该方法可应用于不同年龄或不同大脑区域的小鼠的脑体，研究棕榈醇化的时空结构。

Research

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一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

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科研

生物化学

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Oligopeptide Competition Assay for Phosphorylation Site Determination

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support