Name: quantifying subcellular ubiquitin-proteasome activity in the rodent brain
Uploaded: 2019-05-21T14:30:00
Description: Watch this Scientific Journal Video about Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain at JoVE.com

这项工作得到了农业和生命科学学院和弗吉尼亚理工大学科学学院的启动基金的支持。

所有程序,包括动物科目,都已获得弗吉尼亚理工学院和州立大学动物护理和使用委员会(IACUC)的批准。
1. 啮齿动物脑组织的收集和解剖
注:该协议可应用于各种大脑区域,并用于各种组织采集程序。以下是我们实验室使用8-9周大雄性斯普拉格道利大鼠的鼠脑组织亚细胞程序。为了处理同一动物中的所有细胞隔间,第 1.3 节。无论使用何种组织采集程?...

<ol>
	<li>Hershko, A., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+system.">The ubiquitin system.</a> Annu Rev Biochem. 67, 425-479 (1998).</li><li>Akutsu, M., Dikic, I., Bremm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+chain+diversity+at+a+glance.">Ubiquitin chain diversity at a glance.</a> Journal of Cell Science. 129 (5), 875-880 (2016).</li><li>Ravid, T., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+degradation+signals+in+the+ubiquitin-proteasome+system.">Diversity of degradation signals in the ubiquitin-proteasome system.</a> Nature Reviews Molecular Cell Biology. 9 (9), 679-690 (2008).</li><li>Erpapazoglou, Z., Walker, O., Haguenauer-Tsapis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+roles+of+k63-linked+ubiquitin+chains+in+trafficking.">Versatile roles of k63-linked ubiquitin chains in trafficking.</a> Cells. 3 (4), 1027-1088 (2014).</li><li>Iwai, K., Fujita, H., Sasaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+ubiquitin+chains:+NF-kappaB+signalling,+cell+death+and+beyond.">Linear ubiquitin chains: NF-kappaB signalling, cell death and beyond.</a> Nature Review Molecular Cell Biology. 15 (8), 503-508 (2014).</li><li>Rieser, E., Cordier, S. M., Walczak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+ubiquitination:+a+newly+discovered+regulator+of+cell+signalling.">Linear ubiquitination: a newly discovered regulator of cell signalling.</a> Trends in Biochemical Sciences. 38 (2), 94-102 (2013).</li><li>Collins, G. A., Goldberg, A. 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+Logic+of+the+26S+Proteasome.">The Logic of the 26S Proteasome.</a> Cell. 169 (5), 792-806 (2017).</li><li>Bhat, K. P., 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=The+19S+proteasome+ATPase+Sug1+plays+a+critical+role+in+regulating+MHC+class+II+transcription.">The 19S proteasome ATPase Sug1 plays a critical role in regulating MHC class II transcription.</a> Molecular Immunology. 45 (8), 2214-2224 (2008).</li><li>Ezhkova, E., Tansey, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasomal+ATPases+link+ubiquitylation+of+histone+H2B+to+methylation+of+histone+H3.">Proteasomal ATPases link ubiquitylation of histone H2B to methylation of histone H3.</a> Molecular Cell. 13 (3), 435-442 (2004).</li><li>Jarome, T. J., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+system+as+a+critical+regulator+of+synaptic+plasticity+and+long-term+memory+formation.">The ubiquitin-proteasome system as a critical regulator of synaptic plasticity and long-term memory formation.</a> Neurobiology of Learning and Memory. 105, 107-116 (2013).</li><li>Hegde, A. N., Upadhya, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+pathway+in+health+and+disease+of+the+nervous+system.">The ubiquitin-proteasome pathway in health and disease of the nervous system.</a> Trends in Neuroscience. 30 (11), 587-595 (2007).</li><li>Adori, C., 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=Subcellular+distribution+of+components+of+the+ubiquitin-proteasome+system+in+non-diseased+human+and+rat+brain.">Subcellular distribution of components of the ubiquitin-proteasome system in non-diseased human and rat brain.</a> Journal of Histochemistry and Cytochemistry. 54 (2), 263-267 (2006).</li><li>Upadhya, S. C., Ding, L., Smith, T. K., Hegde, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+proteasome+activity+in+the+nucleus+and+the+synaptic+terminals.">Differential regulation of proteasome activity in the nucleus and the synaptic terminals.</a> Neurochemistry International. 48 (4), 296-305 (2006).</li><li>Enenkel, C., Lehmann, A., Kloetzel, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+distribution+of+proteasomes+implicates+a+major+location+of+protein+degradation+in+the+nuclear+envelope-ER+network+in+yeast.">Subcellular distribution of proteasomes implicates a major location of protein degradation in the nuclear envelope-ER network in yeast.</a> EMBO Journal. 17 (21), 6144-6154 (1998).</li><li>Jarome, T. J., Kwapis, J. L., Ruenzel, W. L., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII,+but+not+protein+kinase+A,+regulates+Rpt6+phosphorylation+and+proteasome+activity+during+the+formation+of+long-term+memories.">CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of long-term memories.</a> Frontiers in Behavioral Neuroscience. 7, 115 (2013).</li><li>Jarome, T. J., Werner, C. T., Kwapis, J. L., Helmstetter, F. J. <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+protein+degradation+is+critical+for+the+formation+and+stability+of+fear+memory+in+the+amygdala.">Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala.</a> PLoS One. 6 (9), e24349 (2011).</li><li>Lopez-Salon, 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=The+ubiquitin-proteasome+cascade+is+required+for+mammalian+long-term+memory+formation.">The ubiquitin-proteasome cascade is required for mammalian long-term memory formation.</a> European Journal of Neuroscience. 14 (11), 1820-1826 (2001).</li><li>Reis, D. S., Jarome, T. J., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+formation+for+trace+fear+conditioning+requires+ubiquitin-proteasome+mediated+protein+degradation+in+the+prefrontal+cortex.">Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.</a> Frontiers in Behavior Neuroscience. 7, 150 (2013).</li><li>Rosenberg, T., Elkobi, A., Rosenblum, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mAChR-dependent+decrease+in+proteasome+activity+in+the+gustatory+cortex+is+necessary+for+novel+taste+learning.">mAChR-dependent decrease in proteasome activity in the gustatory cortex is necessary for novel taste learning.</a> Neurobiology of Learning and Memory. 135, 115-124 (2016).</li><li>Rosenberg, T., Elkobi, A., Dieterich, D. C., Rosenblum, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDAR-dependent+proteasome+activity+in+the+gustatory+cortex+is+necessary+for+conditioned+taste+aversion.">NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion.</a> Neurobiology of Learning and Memory. 130, 7-16 (2016).</li><li>Orsi, S. 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=Distinct+subcellular+changes+in+proteasome+activity+and+linkage-specific+protein+polyubiquitination+in+the+amygdala+during+the+consolidation+and+reconsolidation+of+a+fear+memory.">Distinct subcellular changes in proteasome activity and linkage-specific protein polyubiquitination in the amygdala during the consolidation and reconsolidation of a fear memory.</a> Neurobiology of Learning and Memory. 157, 1-11 (2019).</li><li>Cullen, P. K., Ferrara, N. C., Pullins, S. E., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+memory+formation+requires+activity-dependent+protein+degradation+in+the+hippocampus.">Context memory formation requires activity-dependent protein degradation in the hippocampus.</a> Learning and Memory. 24 (11), 589-596 (2017).</li><li>Jarome, T. J., Ferrara, N. C., Kwapis, J. L., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+regulates+proteasome+phosphorylation+and+activity+and+promotes+memory+destabilization+following+retrieval.">CaMKII regulates proteasome phosphorylation and activity and promotes memory destabilization following retrieval.</a> Neurobiology of Learning and Memory. 128, 103-109 (2016).</li><li>Lee, S. H., 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=Synaptic+protein+degradation+underlies+destabilization+of+retrieved+fear+memory.">Synaptic protein degradation underlies destabilization of retrieved fear memory.</a> Science. 319 (5867), 1253-1256 (2008).</li><li>Dunah, A. W., Standaert, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+D1+receptor-dependent+trafficking+of+striatal+NMDA+glutamate+receptors+to+the+postsynaptic+membrane.">Dopamine D1 receptor-dependent trafficking of striatal NMDA glutamate receptors to the postsynaptic membrane.</a> Journal of Neuroscience. 21 (15), 5546-5558 (2001).</li><li>Kelly, P. T., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+proteins.+Characterization+of+tubulin+and+actin+and+identification+of+a+distinct+postsynaptic+density+polypeptide.">Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide.</a> Journal of Cell Biology. 79 (1), 173-183 (1978).</li><li>Mengual, E., Arizti, P., Rodrigo, J., Gimenez-Amaya, J. M., Castano, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+distribution+and+electron+microscopic+subcellular+localization+of+the+proteasome+in+the+rat+CNS.">Immunohistochemical distribution and electron microscopic subcellular localization of the proteasome in the rat CNS.</a> Journal of Neuroscience. 16 (20), 6331-6341 (1996).</li></ol>

在这里,我们演示了一种有效的方法,用于量化同一动物中不同亚细胞腔中泛基蛋白酶体活性的变化。目前,测量泛基蛋白蛋白酶体系统活性亚细胞变化的大多数尝试被限制在每个样品的单个隔间内,因此需要重复实验。这会导致动物生命的巨大成本和损失。我们的协议通过分裂半球来缓解这个问题,允许从同一动物的每个半球收集不同的细胞分数。使用这个协议,我们能够首次证明,在同一动物泛素-蛋?...

泛性蛋白-蛋白酶体系统(UPS)是一个复杂的网络,由相互连接的蛋白质结构和气体组成,控制着细胞1中大多数短寿命蛋白质的降解。在这个系统中,蛋白质被小修饰剂泛性蛋白标记为降解或其他细胞过程/命运。靶蛋白可以获得1-7种泛基质修饰,它可以在7个流氨酸(K11、K27、K29、K33、K48和K63)或N端蛋氨酸(M1;称为线性)中的七个基氨酸(K)位点之一连接在一起。其中一些多维他金标记是降解特异性(K48)3,而其他基本上与蛋白质降解过程(M1)4,5,6无关。因此,蛋白质泛化过程极其复杂,量化特定多聚素标记的变化的能力对于最终理解这种给定修饰在细胞功能中的作用至关重要。进一步复杂化的研究这个系统,蛋白酶体,这是UPS7的催化结构,既降解蛋白质,但也可以参与其他非蛋白酶过程8,9。毫不奇怪,自最初发现以来,正常和异常的泛性蛋白-蛋白酶体活动一直与长期记忆形成和各种疾病状态有关,包括许多神经、神经退行性和精神病紊乱10,11。因此,能够有效和高效地量化UPS在大脑中的活动的方法对于最终理解这个系统在疾病状态下是如何调节不良以及最终开发针对泛基丁和/或的治疗方案至关重要。蛋白酶体功能。
在量化大鼠和小鼠脑组织中泛基蛋白酶体活性方面存在许多问题,这是用于研究UPS功能的最常见模型系统,包括1)泛基蛋白修饰的多样性,以及2)分布和UPS在亚细胞舱12、13、14之间运作的差分调节。例如,在记忆形成过程中大脑中泛素蛋白酶体功能的许多早期演示使用全细胞赖沙,并表明蛋白质泛化和蛋白酶体活性15的时间依赖性增加,16,17,18,19,20.然而,我们最近发现,泛基蛋白-蛋白酶体活性在亚细胞间中因学习而差异很大,有些区域同时增加,另一些区域减少,这一模式差异很大从之前报告在整个细胞莱沙21。这与整个细胞方法的限制是一致的,因为它不能分离不同亚细胞隔间UPS活动的变化。虽然最近的研究已经使用突触分数协议,以研究UPS专门在突触响应学习22,23,24,使用的方法遮挡了测量的能力同一动物的核质和细胞质泛素蛋白体变化。这导致不必要的需要重复实验多次,收集不同的亚细胞分数在每个。这不仅会导致动物生命损失,而且消除了直接比较不同亚细胞间的UPS活动以响应给定事件或特定疾病状态的能力。考虑到泛素蛋白和蛋白酶体的蛋白质靶点在整个细胞中差异很大,了解泛素蛋白-蛋白酶体信号在不同亚细胞室中如何不同,对于确定UPS在细胞中的功能作用至关重要。大脑在记忆形成和神经,神经退行性和精神障碍。
为了满足这一需求,我们最近开发了一个程序,其中核,细胞质和突触分数可以收集从同一动物21的给定大脑区域。此外,为了考虑从同一样品中收集多个亚细胞分数可以获得的有限蛋白质量,我们优化了先前建立的协议,以检测体外蛋白酶体活性和联动特异性从啮齿动物脑组织收集的分片细胞中的蛋白质普遍化。使用该协议,我们能够收集和直接比较蛋白酶体活性、K48、K63、M1和细胞质中整体蛋白多聚体化水平以及大鼠侧杏仁核的突合体中学习相关变化。在这里,我们详细介绍了我们的程序(图1),这可以显著提高我们对UPS如何参与长期记忆形成和各种疾病状态的理解。然而,应该指出的是,我们的协议中讨论的体外蛋白酶体活性虽然被广泛使用,但并不能直接测量完整的26S蛋白酶体复合物的活性。相反,此检测测量 20S 内核的活动,这意味着它只能作为代理来了解内核本身的活动,而不是整个 26S 蛋白酶体复合体。

<table border="0" cellpadding="0" cellspacing="0" style="width:878px;" width="878">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">0.5M EDTA</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:259px;">15575020</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">20S Proteasome Activity Kit</td>
			<td>Millipore Sigma</td>
			<td>APT280</td>
			<td>Other vendors carry different versions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">ATP</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:259px;">FERR1441</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Beta-actin antibody</td>
			<td>Cell signaling</td>
			<td>4967S</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Beta-tubulin antibody</td>
			<td>Cell signaling</td>
			<td>2128T</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BioTek Synergy H1 plate reader</td>
			<td>BioTek</td>
			<td>VATECHH1MT3</td>
			<td>Other vendors carry different versions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B-mercaptoethanol</td>
			<td>Fisher</td>
			<td>ICN19024280</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">clasto lactacystin b-lactone</td>
			<td>Millipore Sigma</td>
			<td>L7035</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cryogenic cup</td>
			<td>Fisher</td>
			<td>033377B</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMSO</td>
			<td>DMSO</td>
			<td>D8418</td>
			<td>Varous other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">DTT</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">D0632</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">Glycerol</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">G5516</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">H3 antibody</td>
			<td>Abcam</td>
			<td>ab1791</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">HEPES</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">H3375</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hydrochloric acid</td>
			<td>Fisher</td>
			<td>SA48</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">IGEPAL (NP-40)</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">I3021</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">K48 Ubiquitin Antibody</td>
			<td>Abcam</td>
			<td>ab140601</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">K63 Ubiquitin Antibody</td>
			<td>Abcam</td>
			<td>ab179434</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">KCl</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">P9541</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KONTES tissue grinder</td>
			<td>VWR</td>
			<td>KT885300-0002</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laemmli sample buffer</td>
			<td>Bio-rad</td>
			<td>161-0737</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Linear Ubiquitin Antibody</td>
			<td>Life Sensors</td>
			<td>AB-0130-0100</td>
			<td>Only M1 antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">MgCl</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">442611</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>2231000213</td>
			<td>Various other manufacturers/models</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">myr-AIP</td>
			<td>Enzo Life Sciences</td>
			<td>BML-P212-0500</td>
			<td>Carried by Millipore-Sigma</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">NaCl</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">S3014</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Odyssey Fc Imaging System</td>
			<td>LiCor</td>
			<td>2800-02</td>
			<td>Other vendors carry different versions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">Phosphatase Inhibitor</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">524625</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Precision Plus Protein Standard</td>
			<td>Bio-rad</td>
			<td>161-0373</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">Protease Inhibitor</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">P8340</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PSD95 antibody</td>
			<td>Cell signaling</td>
			<td>3450T</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:222px;">SDS</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">L3771</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:46px;">Sodium hydroxide</td>
			<td>Fisher</td>
			<td>SS255</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:222px;">Sucrose</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">S0389</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">TBS</td>
			<td>Alfa Aesar</td>
			<td>J62938</td>
			<td>Varous other vendors</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:50px;width:222px;">Tris</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">T1503</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Tween-20</td>
			<td>Fisher</td>
			<td>BP337-100</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Ubiquitin Antibody</td>
			<td>Enzo Life Sciences</td>
			<td>BML-PW8810</td>
			<td>Various other vendors</td>
		</tr>
	</tbody>
</table>

quantifying subcellular ubiquitin-proteasome activity in the rodent brain

泛素蛋白体系统是真核生物中蛋白质降解和各种其他细胞过程的关键调节器。在大脑中,泛英蛋白酶体活性的增加对突触可塑性至关重要,记忆形成和本系统中的异常变化与各种神经、神经退行性和精神障碍有关。研究大脑中泛泛蛋白蛋白酶功能的一个问题是,它存在于所有细胞隔间中,其中蛋白质靶点、功能作用和调节机制可能有很大差异。因此,能够直接比较同一动物内不同亚细胞室中大脑泛性蛋白靶向和蛋白酶体催化活性的能力,对于充分理解UPS如何促进突触可塑性至关重要,记忆和疾病。此处描述的方法允许从同一啮齿动物(大鼠)大脑中收集核、细胞质和粗突触分数,然后同时定量蛋白酶体催化活性(间接提供蛋白酶体核心的活性)仅)和连杆特异性泛基蛋白标记。因此,该方法可用于直接比较同一动物在突触可塑性、记忆形成和不同疾病状态期间不同大脑区域泛性蛋白酶体活性的亚细胞变化。该方法还可用于评估同一动物中其他蛋白质的亚细胞分布和功能。

使用此处描述的程序,从大鼠大脑的侧杏仁核中收集核、细胞质和突触分数(图1)。单个馏分的纯度通过西方印迹得到确认,用抗体对应富于或耗尽的莱沙中的蛋白质进行抗体探测。在收集粗突触分数的第一个半球,突触密度蛋白95(PSD95)存在于突触中,但不是核,细胞质水平较低(图2A)。这与先前的工作一致,表明突触分数制剂分离了前突...

Watch this Scientific Journal Video about Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain at JoVE.com

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

该协议旨在有效地量化啮齿动物大脑不同细胞舱中的泛泛蛋白-蛋白酶体系统(UPS)活性。用户能够检查UPS在同一动物中核、细胞质和突触分数的功能,从而减少执行这些复杂分析所需的时间和动物数量。

量化龙脑中的亚细胞泛基蛋白-蛋白酶体活性

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...

该协议测量亚细胞蛋白泛化水平和啮齿动物大脑中蛋白酶活性，允许在受试者内部比较泛素蛋白酶体活动如何随细胞活动、学习或疾病而变化。该协议允许从同一啮齿动物大脑收集突触、细胞质和核分数。最大限度地减少损失，它可以通过小组织量和基本的实验室设备进行。证明这个程序的将是泰勒·麦克法登，一个研究生从我的实验室。开始这个程序与收集和解剖啮齿动物脑组织，如文本协议中所述。确保使用的半球在每个实验组的提取条件下保持平衡。从零下80摄氏度的冰柜中取出含有一个半球的1.5毫升离心式微管。使用手术刀，将冷冻脑组织转移到两毫升玻璃特氟龙均质器。向特氟隆管中加入500微升的解液缓冲液。使用 pestle B，用 15 次冲程使同一组织均质，直到不存在可见数量的固体物质。在每个冲程期间使用转弯运动。使用 1，000 微升移液器，将均质样品转移到新的 1.5 毫升微离心管中。将管子放在湿冰上孵育30分钟。将管子放在微离心中，在4摄氏度的845倍g下旋转10分钟。完成后，通过移液小心地取出上一液，并放入新的1.5毫升微离心管中。这是细胞质分数。将 50 微升萃取缓冲液添加到产生的颗粒中，然后通过移液重新暂停。不要涡旋颗粒。将含有再增颗粒的管子放在冰上孵育30分钟。然后在4摄氏度的21，456次g下将管离心20分钟。离心后，通过移液小心去除上经剂，并放入新的1.5毫升微离心管中。这是核部分。与以前一样，将感兴趣区域的一个半球同质化，除了使用 500 微升的 TEVP 缓冲液而不是解解缓冲液。使用1000微升移液器，将均质样品转移到新的1.5毫升微离心管中。将样品以1000倍g离心，在4摄氏度下10分钟。收集上流水液，然后使用1000微升移液器将其转移到新的1.5毫升微离心管中。在4摄氏度下，以10，000倍g离心样品10分钟。丢弃含有核和大碎屑的原始颗粒。将上流液转移到新的 1.5 毫升微离心管中。这是细胞分位。在颗粒中加入50微升均质缓冲液，然后通过移液重新暂停，直到看不到固体材料。将样品以20，000倍g离心，在4摄氏度下10分钟。将上流液转移到新的 1.5 毫升微离心管中。这是粗突触体膜分数。要设置测定，将板读卡器预热至37摄氏度，并按住运行。将激发设置为 360 纳米，将发射量设置为 460 纳米。如果使用的 96 井板清晰，请将光学位置设置为底部。如果使用深色 96 井板，请将光学位置设置为顶部。对动能运行进行编程，每 30 分钟扫描两小时。将套件中提供的 20S 10X 检测缓冲液与 13.5 毫升超纯水重组。将 14 微升 100 毫摩尔 ATP 添加到现在的 1X 缓冲器中。这显著增强了样品中的蛋白酶体活性，并提高了测定的可靠性。重组套件中提供的 AMC 标准，并配有 100 微升的 DMSO。在黑暗或低光条件下使用 AMC 标准执行步骤，因为该标准对光敏感。创建从一系列高到低AMC浓度的AMC标准曲线。此曲线将用于板读卡器校准和分析均质样品中的蛋白酶体活性。用 65 微升的 DMSO 试剂盒重建套件中提供的蛋白酶体子。由于基板对光敏感，因此在黑暗或低光条件下执行此步骤。然后使用20S测定缓冲液，在新的1.5毫升微离心管中产生1至20个蛋白酶体基质的稀释。将所需样品的规范化量添加到 96 井板中。在重复项中运行每个示例。所需的样品量因组织制备而异。通常，10 到 20 微克对于任何亚细胞分数都足够。用超纯水将样品井体积调至 80 微升。添加的数量取决于添加的样本量。在两个独立的井中，仅增加80微升水。这些将是检测空白。在每个井中加入10个20S检测缓冲液，包括检测空白。使用中继器或自动移液器，确保孔的检测量一致。关灯或进入黑暗的房间。将所有 100 微升稀释的 AMC 标准添加到新井中，每个标准使用一个井。在黑暗中，使用中继器或自动移液器将10微升稀释蛋白酶体基板添加到含有样品和测定空白但不包括AMC标准的井中。将板放入板读卡器中，然后开始动动运行。使用各种标准西方印迹方案，结合独特的联动特异性多泛素抗体，对从啮齿动物脑组织收集的不同亚细胞分数中的各种多泛素标记进行定量。一些泛素的西方印迹图像将提供不同波段的列，而另一些则生成具有很少或没有清晰线条的污迹图案。为了量化泛素西方印迹，在柱子周围画一个框，延伸整个分子标准阶梯。如果泛素染色确实延伸到整个梯子，请调整包装盒。这在 Lysine 48 修改中很常见，并且在亚细胞隔间之间差别很大。最后，减去背景，该背景计算为背景在各侧立即围绕列的均光密度。这里展示的是从同一动物的横向杏仁核收集的不同分数中蛋白酶体活性的定量。在体外蛋白酶体活性测定中，检测到的突触分数、细胞质分数和核分的检测从开始到结束的相对荧光单位增加。蛋白酶体抑制剂β-lac阻止R2在整个会话中变化。在同一动物的横向杏仁核中观察到蛋白酶体活性的亚细胞差异。在训练有素的动物中检测到核蛋白酶体活性增加，这与突触分数内活动减少相对应。细胞质蛋白酶体活性保持在基线。这里展示的是同一动物学习后在横向杏仁核中连结特异性蛋白质泛化的亚细胞差异。学习后，核分数的整体泛化率增加，这与细胞质分数的减少有关。学习后，线性泛化增加在核分数，但不是细胞质或突触分数。有趣的是，K63泛化在学习后在核分数中增加，这与突触分数的减少有关。而K48单体在学习后在核和细胞质分数中增加，但在突触分数中则不增加。该协议还可用于了解同一动物中其他蛋白质的亚细胞分布和功能。

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Unit 1

Translation: RNA to Protein

SCIENTISTS IN ACTION

Simultaneous fMRI and Electrophysiology in the Rodent Brain

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Personalized Needles for Microinjections in the Rodent Brain

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...