The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

以下协议符合动物保护的加拿大议会的指引，并已获得医药动物保健委员会的教授在多伦多大学。 1，准备好所需的试剂中的说明表1 <ol><li>添加蛋白酶和磷酸酶（如果需要的话）的抑制剂，以所有的蔗糖溶液，缓冲液和DDH 2 O的以表1所列的浓度。重要：执行所有的步骤在4℃和预冷所有在实验前的起始试剂和设备。标签所有管道，包括超速离心管，用记号笔。 </li></ol> 2，解剖相应的脑区<ol><li>颈椎脱位或断头牺牲动物。众议院和安乐死的动物，根据动物保健机构和政府的政策。 </li><li>快速去除日ê大脑从冷冻解剖块头骨和地点。 </li><li>从新鲜组织解剖相应的大脑区域，并继续执行步骤3。 </li></ol> 3，组织匀浆用电机驱动玻璃特氟隆均质机<ol><li>将解剖组织50-100毫克（为SPM）或100-200毫克（为PSD），在含有4毫升的0.32M HEPES缓冲蔗糖溶液标记13毫升聚丙烯管。将样品转移到15毫升锥形玻璃铁氟龙组织研磨机/均质器，设置电机驱动器900转（设置7），均质12招在30秒时段的样本。使用不同的玻璃匀浆器特氟龙样品之间，或用清水冲洗样本之间的冷蒸馏水的均质机，用的Kimwipe擦干。注：最多4个克组织的可匀浆于4毫升的0.32M HEPES缓冲蔗糖溶液，但最好是使用较少的组织，以确保适当的分级分离。 </li><li>均匀化的样本传送回吨Ø同13毫升聚丙烯管。储备100微升匀浆并存储在-80℃用于随后的蛋白质定量和总蛋白级分的免疫印迹分析。 </li></ol> 4，低速离心去除细胞核部分（收益率上清S1） <ol><li>离心机在4℃下将匀浆物在一个固定角转子，在900×g离心10分钟。上清液（S1）转移到一个新的，标为13毫升管和悬浮细胞核部分沉淀（P1），在500微升的0.32M HEPES缓冲蔗糖。注：（P1）的部分可被存储在-80℃下，并用于核级分的免疫印迹分析。 </li></ol> 5，丰富的粗突触体部分的（球团产量P2） <ol><li>离心上清液（S1）以10,000 xg离心15分钟，在4℃下。取出上清液（S2）和储备500μL的微量离心管中保存在-80℃，随后蛋白定量ification和胞浆/轻膜部分免疫印迹分析。重悬剩余的粗突触体部分沉淀（P2）在1ml的0.32M HEPES缓冲蔗糖溶液，然后添加另一毫升0.32的3M HEPES缓冲蔗糖溶液。 </li><li>离心机以10,000×g进行15分钟，在4℃下。取出上清液（S2&#39;），并储备500μL的微量离心管中保存在-80℃，随后蛋白定量和胞浆/轻膜部分免疫印迹分析。保存洗涤的粗突触体丸粒（P2&#39;）的聚丙烯管中。 </li></ol>在原油突触体部分的6裂解（低渗休克） <ol><li>裂解的粗突触体丸粒（P2&#39;）的聚丙烯管中再悬浮是在1ml的DDH 2 O的添加另一个3毫升DDH 2 O的，转移到玻璃铁氟龙组织匀浆，并经均质手3招。注意：这是很重要的以尽可能快地执行此步骤，并迅速进行到步骤6.2。 </li><li>从均化器放回同一13毫升聚丙烯管迅速转移样品调整样本回至4毫米的HEPES和16微升的1M HEPES溶液，颠倒混合。 </li><li>转动样品，在4℃下进行30分钟以确保完全裂解。 </li></ol> 7，丰富的突触膜部分（P3） <ol><li>离心机以25,000×g离心20分钟，在4℃下。除去粗泡状部分上清液（S3），并保留其在5毫升管存放于-80℃，随后蛋白定量和Western blot分析。重悬的突触体膜部分（P3）在1ml的0.32M HEPES缓冲蔗糖溶液。 </li></ol> 8，制备连续蔗糖梯度<ol><li>吸管准确3.5毫升的1.2M HEPES缓冲蔗糖溶液和准确3.0毫升每1.0摩尔和0.8摩尔HEPES-缓冲蔗糖溶液为单独6毫升卡口盖管。注意：这是为了预先测量，用于使蔗糖梯度的缓冲液的体积。卷的精确测量是非常重要的，以确保所得到的梯度将在超速离心平衡。 </li><li>使用玻璃巴斯德吸管和灯泡，转移的1.2M HEPES缓冲蔗糖溶液的12毫升异质同超速离心管。小心层上的1.2M HEPES缓冲蔗糖溶液的顶部的1.0M HEPES缓冲蔗糖溶液。最后，层上的1.0M HEPES缓冲蔗糖溶液的顶部的0.8M的HEPES缓冲蔗糖溶液。使用新鲜的巴斯德吸管每个蔗糖溶液，并注意不要打扰蔗糖梯度。从板凳漩涡或离心振动会干扰梯度的完整性。 </li><li>使用巴斯德吸管，层上的准备连续蔗糖梯度顶部的悬浮突触膜部分（P3）。确保该梯度是通过称量它们的水平​​转头的水桶内平衡。如果水桶是不均衡的，使用P200移液管小心地添加的0.32M HEPES缓冲蔗糖梯度的顶部和平衡转子轮叶。 </li></ol> 9分馏的突触质膜（SPM） <ol><li>超速离心机的水平转头150000 XG 2小时，4℃。小心地从超速离心桶取出蔗糖梯度。用18G的针和1ml注射器，穿刺管在1.0米/ 1.2M的HEPES缓冲蔗糖溶液相间的底部，并撤回突触质膜层（SPM）。 </li><li>记每个SPM样品占据的1ml注射器中的体积。将收集到的SPM层在3.5mL厚壁超速离心管中，添加恰好2.5体积的4 mM的HEPES调节从1.2M的蔗糖浓度为0.32 M。一旦各样品已经调整编到0.32M蔗糖，平衡管和0.32M的HEPES缓冲蔗糖溶液。 </li><li>超速离心机在一个固定角转头200000×g离心30分钟，在4℃。拆下并丢弃上清液。悬浮突触质膜颗粒（SPM）的300微升50毫米肝素钠/ 2毫米的EDTA溶液。直到用于免疫印迹商店的样品在-80℃。 </li></ol> 10，准备突触后密度的分数（PSD） <ol><li>解冻的SPM样品在冰上。 </li><li>让0.54％的Triton X-100的50毫米肝素钠/ 2毫米EDTA溶液的解决方案。 </li><li>在5ml的聚苯乙烯管中，结合2.7的Triton X-100 / HEPES / EDTA溶液和300μl的SPM分数毫升，旋转样品15分钟，在4℃下</li><li>转移样品至3.5毫升厚壁超速离心管并离心样品在32,000 xg离心20分钟，在4℃下。 </li><li>保留上清液（曲拉通X-100可溶组分[TS]）​​，并储存在-80;°℃。注：在此阶段，将沉淀表示“PSD-1T”分数。用洗衣粉第二次治疗将产生一个“PSD-2T”部分。如果PSD-1T部分需要，继续执行步骤10.6。 <ol><li>以产生一个PSD-2T馏分，重悬在3ml 0.5％的Triton粒料X-100的50mM HEPES / 2mM EDTA的溶液中。旋转样品15分钟，在4℃下。以32,000×g离心20分钟，在4℃下离心样品，并继续执行步骤10.6。 </li></ol></li><li>弃上清，重悬突触后（PSD）颗粒在50毫米肝素钠/ 2 mM的EDTA溶液。注：该悬浮量可取决于颗粒的大小;建议50-75微升的体积。由于PSD的分数是洗涤剂不溶部分，通常需要加入1-2微升0.5％SDS中以使蛋白质的完整的再悬浮。直到用于免疫印迹商店的样品在-80℃。 </li></ol>

<ol>
	<li>Delint-Ramirez, 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=In+vivo+composition+of+NMDA+receptor+signaling+complexes+differs+between+membrane+subdomains+and+is+modulated+by+PSD-95+and+PSD-93.">In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 30, 8162-8170 (2010).</li><li>Wang, Q., 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+psychiatric+disease+risk+factors+DISC1+and+TNIK+interact+to+regulate+synapse+composition+and+function.">The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function.</a> Mol Psychiatry. 16, (2010).</li><li>Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinsertion+or+degradation+of+AMPA+receptors+determined+by+activity-dependent+endocytic+sorting.">Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting.</a> Neuron. 28, 511-525 (2000).</li><li>Tomita, S., Fukata, M., Nicoll, R. A., Bredt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+interaction+of+stargazin-like+TARPs+with+cycling+AMPA+receptors+at+synapses.">Dynamic interaction of stargazin-like TARPs with cycling AMPA receptors at synapses.</a> Science. 303, 1508-1511 (2004).</li><li>Ehlers, M. D., Heine, M., Groc, L., Lee, M. C., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusional+trapping+of+GluR1+AMPA+receptors+by+input-specific+synaptic+activity.">Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity.</a> Neuron. 54, 447-460 (2007).</li><li>Bats, C., Groc, L., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+Stargazin+and+PSD-95+regulates+AMPA+receptor+surface+trafficking.">The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking.</a> Neuron. 53, 719-734 (2007).</li><li>Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+level+controls+postsynaptic+composition+and+signaling+via+the+ubiquitin-proteasome+system.">Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system.</a> Nat Neurosci. 6, 231-242 (2003).</li><li>Cartier, E. 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=A+biochemical+and+functional+protein+complex+involving+dopamine+synthesis+and+transport+into+synaptic+vesicles.">A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles.</a> J Biol Chem. 285, 1957-1966 (2010).</li><li>Rodriguez de Lores, A., Alberici, M., De Robertis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+and+enzymic+studies+of+cholinergic+and+non-cholinergic+synaptic+membranes+isolated+from+brain+cortex.">Ultrastructural and enzymic studies of cholinergic and non-cholinergic synaptic membranes isolated from brain cortex.</a> Journal of Neurochemistry. 14, 215-225 (1967).</li><li>Gray, E. G., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+nerve+endings+from+brain:+an+electron-microscopic+study+of+cell+fragments+derived+by+homogenization+and+centrifugation.">The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation.</a> J Anat. 96, 79-88 (1962).</li><li>Cotman, C. W., Taylor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structural+studies+on+synaptic+complexes+from+rat+brain.">Isolation and structural studies on synaptic complexes from rat brain.</a> J Cell Biol. 55, 696-711 (1972).</li><li>Cotman, C. W., Banker, G., Churchill, L., Taylor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+postsynaptic+densities+from+rat+brain.">Isolation of postsynaptic densities from rat brain.</a> The Journal of Cell Biology. 63, 441-455 (1974).</li><li>Carlin, R. K., Grab, D. J., Cohen, R. S., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+postsynaptic+densities+from+various+brain+regions:+enrichment+of+different+types+of+postsynaptic+densities.">Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.</a> The Journal of Cell Biology. 86, 831-845 (1980).</li><li>Jones, D. H., Matus, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+synaptic+plasma+membrane+from+brain+by+combined+flotation-sedimentation+density+gradient+centrifugation.">Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation.</a> Biochim Biophys Acta. 356, 276-287 (1974).</li><li>Kennedy, M. B., Bennett, M. K., Erondu, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+immunochemical+evidence+that+the+"major+postsynaptic+density+protein"+is+a+subunit+of+a+calmodulin-dependent+protein+kinase.">Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 80, 7357-7361 (1983).</li><li>Moon, I. S., Apperson, M. L., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+tyrosine-phosphorylated+protein+in+the+postsynaptic+density+fraction+is+N-methyl-D-aspartate+receptor+subunit+2B.">The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 3954-3958 (1994).</li><li>Cho, K. O., Hunt, C. A., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+brain+postsynaptic+density+fraction+contains+a+homolog+of+the+Drosophila+discs-large+tumor+suppressor+protein.">The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.</a> Neuron. 9, 929-942 (1992).</li><li>Salahpour, 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=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc Natl Acad Sci U S A. 105, 4405-4410 (2008).</li><li>Ma, D. K., 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=Neuronal+activity-induced+Gadd45b+promotes+epigenetic+DNA+demethylation+and+adult+neurogenesis.">Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis.</a> Science. 323, 1074-1077 (2009).</li><li>Budreck, E. C., Scheiffele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin-3+is+a+neuronal+adhesion+protein+at+GABAergic+and+glutamatergic+synapses.">Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses.</a> The European Journal of Neuroscience. 26, 1738-1748 (2007).</li><li>Wu, 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=Arc/Arg3.1+regulates+an+endosomal+pathway+essential+for+activity-dependent+beta-amyloid+generation.">Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent beta-amyloid generation.</a> Cell. 147, 615-628 (2011).</li><li>Huttner, W. B., Schiebler, W., Greengard, P., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsin+I+(protein+I),+a+nerve+terminal-specific+phosphoprotein.+III.+Its+association+with+synaptic+vesicles+studied+in+a+highly+purified+synaptic+vesicle+preparation.">Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation.</a> J Cell Biol. 96, 1374-1388 (1983).</li><li>Nagy, A., Baker, R. R., Morris, S. J., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preparation+and+characterization+of+synaptic+vesicles+of+high+purity.">The preparation and characterization of synaptic vesicles of high purity.</a> Brain Res. 109, 285-309 (1976).</li><li>Cheng, D., 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=Relative+and+absolute+quantification+of+postsynaptic+density+proteome+isolated+from+rat+forebrain+and+cerebellum.">Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.</a> Molecular &amp; Cellular Proteomics : MCP. 5, 1158-1170 (2006).</li><li>Sheng, M., Hoogenraad, C. 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+postsynaptic+architecture+of+excitatory+synapses:+a+more+quantitative+view.">The postsynaptic architecture of excitatory synapses: a more quantitative view.</a> Annu Rev Biochem. 76, 823-847 (2007).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a Laboratory Manual. , (1989).</li><li>Eggena, 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=Identification+of+histone+H1+as+a+cognate+antigen+of+the+ulcerative+colitis-associated+marker+antibody+pANCA.">Identification of histone H1 as a cognate antigen of the ulcerative colitis-associated marker antibody pANCA.</a> J Autoimmun. 14, 83-97 (2000).</li><li>Salahpour, 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=Homodimerization+of+the+beta2-adrenergic+receptor+as+a+prerequisite+for+cell+surface+targeting.">Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting.</a> J Biol Chem. 279, 33390-33397 (2004).</li><li>Bernocco, S., 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=Sequential+detergent+fractionation+of+primary+neurons+for+proteomics+studies.">Sequential detergent fractionation of primary neurons for proteomics studies.</a> Proteomics. 8, 930-938 (2008).</li><li>Grunewald, T. 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=Nuclear+localization+and+cytosolic+overexpression+of+LASP-1+correlates+with+tumor+size+and+nodal-positivity+of+human+breast+carcinoma.">Nuclear localization and cytosolic overexpression of LASP-1 correlates with tumor size and nodal-positivity of human breast carcinoma.</a> BMC Cancer. 7, 198 (2007).</li><li>Heimann, K., Percival, J. M., Weinberger, R., Gunning, P., Stow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+isoforms+of+actin-binding+proteins+on+distinct+populations+of+Golgi-derived+vesicles.">Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles.</a> J Biol Chem. 274, 10743-10750 (1999).</li><li>Neff, R. A., Gomez-Varela, D., Fernandes, C. C., Berg, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+scaffolds+for+nicotinic+receptors+on+neurons.">Postsynaptic scaffolds for nicotinic receptors on neurons.</a> Acta Pharmacol Sin. 30, 694-701 (2009).</li><li>Cella, N., Cornejo-Uribe, R. R., Montes, G. S., Hynes, N. E., Chammas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lysosomal-associated+membrane+protein+LAMP-1+is+a+novel+differentiation+marker+for+HC11+mouse+mammary+epithelial+cells.">The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells.</a> Differentiation. 61, 113-120 (1996).</li><li>Otera, 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=Peroxisomal+targeting+signal+receptor+Pex5p+interacts+with+cargoes+and+import+machinery+components+in+a+spatiotemporally+differentiated+manner:+conserved+Pex5p+WXXXF/Y+motifs+are+critical+for+matrix+protein+import.">Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import.</a> Mol Cell Biol. 22, 1639-1655 (2002).</li><li>Goubaeva, F., 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=Cardiac+mitochondrial+connexin+43+regulates+apoptosis.">Cardiac mitochondrial connexin 43 regulates apoptosis.</a> Biochem Biophys Res Commun. 352, 97-103 (2007).</li><li>Lamers, K. 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=Protein+S-100B,+neuron-specific+enolase+(NSE),+myelin+basic+protein+(MBP)+and+glial+fibrillary+acidic+protein+(GFAP)+in+cerebrospinal+fluid+(CSF)+and+blood+of+neurological+patients.">Protein S-100B, neuron-specific enolase (NSE), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) and blood of neurological patients.</a> Brain Res Bull. 61, 261-264 (2003).</li><li>Arunachalam, 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=Munc18-1+is+critical+for+plasma+membrane+localization+of+syntaxin1+but+not+of+SNAP-25+in+PC12+cells.">Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells.</a> Mol Biol Cell. 19, 722-734 (2008).</li><li>Brandstatter, J. H., Fletcher, E. L., Garner, C. C., Gundelfinger, E. D., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+presynaptic+cytomatrix+protein+bassoon+among+ribbon+synapses+in+the+mammalian+retina.">Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina.</a> Eur J Neurosci. 11, 3683-3693 (1999).</li></ol>

The authors have no conflicts of interest to disclose.

还有在该过程的几个步骤，这对于一个成功的结果至关重要。在步骤3中，重要的是均质化的一致度，实现对每个样品。当均质化组织与马达驱动的均化器，以恒定速度不仅用于杵的旋转，而且还与笔划的数量。在渗透休克的温育时间应精确，因为在低渗溶液中的扩展同质或孵化将裂解线粒体和污染SPM样品。 另一个关键步骤是在试剂，尤其是蔗糖溶液的制备;如果该蔗糖溶液的...

神经元通过突触进行通信，并且该通信质量是通过在蛋白质在突触的组成改​​变调节到很大程度。特别是位于突触后密度蛋白通过密切脚手架神经递质受体与它们的信号转导系统1参与神经元沟通。此外，在突触效能的强度持久的变化是由于突触后密度1-6添加或去除受体的控制。因此，突触蛋白的分离和定量是一种必要的和有用的技术，来获得有关该神经元响应的刺激和改变突触效能7的方式。本文介绍了一种常见的技术，通过离心分离从啮齿动物脑组织中突触蛋白在连续蔗糖梯度。突触细胞膜部分可以得到丰富和孤立的基础上其在蔗糖密度，已根据经验确定，以类似于1.2M的蔗糖。 根据不同的生物学问题，亚细胞组分可以由蔗糖或珀可连续或不连续梯度进行分离。连续梯度允许蛋白质成多个组分的分离;这可以是特别有用的，以证明一个给定的级分8中的共定位的蛋白质。然而，连续梯度的制备是更费力的和是不必要的许多应用。不连续的梯度是比较容易制备和可用于蛋白分离成几，通常定义的馏分。这是由增加的摩尔浓度的3蔗糖层的不连续梯度，已被广泛用于分离与突触质膜（SPM）相关的蛋白质。本突触质膜级分可被进一步加工，以突触后密度fractioN（PSD）的洗涤剂不溶部分的去污剂处理和隔离。 当这个过程在1960年的9,10首次被描述，电子显微镜被用来证实细胞器及膜的大致限定突触质膜和突触后密度级分9-14。这些研究证明了包含前和突触后膜和在SPM分数突触小泡;洗涤剂治疗主要是电子致密后，突触后密度为可见的。在该过程中，低渗休克用于从细胞体10夹断一样处理。这个步骤需要的事实，线粒体是渗透冲击更耐和保持不动，并且因此它们在蔗糖梯度（ 图1）的底部沉淀的优点。 用同样的富集技术，对SPM和PSD分数分别为第一生化通过聚丙烯酰胺凝胶电泳和主要蛋白质组分15-17的序列来定义。随后免疫印迹分析已被用于检测和定量突触蛋白的水平，并进一步确定这些级分（ 图2）。我们已经使用该技术在我们的实验室进行量化改变多巴胺转运时Slc6a3基因被复制在小鼠18所发生的突触水平。我们也使用这种技术在NMDA受体缺陷小鼠揭示突触特异性降低的蛋白质，是在DISC1相互作用组19的一部分。 它是由免疫印迹分析了SPM的馏分含有突触小泡膜蛋白，核内体标记物，线粒体蛋白质，膜相关合成酶和信号转导分子，以及突触后密度的组成部分和突触质膜20-23明显。 ËVEN PSD级分可具有污染具有丰富的线粒体蛋白质，它可能有必要进行第二梯度沉降或额外的纯化步骤，以除去它们13。最近，定量质谱法提供了超过100个蛋白质在单独的突触后密度的列表，以及这些部件24,25的相对丰度的指示。

<table border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">1M HEPES, pH 7.4</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">HEP003.100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">HEPES</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">HEP001.500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sucrose&#160;</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SUC507.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">EDTA</td>
			<td style="width:129px;">BioBasic</td>
			<td style="width:111px;">EB0185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">PMSF</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">PMS123.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Aprotinin</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">APR600.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Leupeptin</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">LEU001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Pepstatin</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">PEP605.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Benzamidine</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">BEN601.25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sodium fluoride</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SFL001.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sodium pyrophosphate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SPP310.100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Sodium orthovanadate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SOV664.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">&#946;-glycerophosphate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">GYP001.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Triton X-100</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">TRX506.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">18G x 1 &#189;&#8221; needle</td>
			<td style="width:129px;">BD</td>
			<td style="width:111px;">305196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">1cc syringe</td>
			<td style="width:129px;">BD</td>
			<td style="width:111px;">309659</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;"></td>
			<td style="width:129px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Equipment</td>
			<td style="width:129px;">Company</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Glass Teflon homogenizer Kontes Duall 23</td>
			<td style="width:129px;">VWR</td>
			<td style="width:111px;">KT885450-0023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">IKA Model RW 16 Basic Stirrer</td>
			<td style="width:129px;">IKA Works</td>
			<td style="width:111px;">2572100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall SM-24 Fixed Angle Rotor</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">29017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall RC 6 Plus Centrifuge</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">46910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thinwall Polyallomer Tubes (13.2mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SW 41 Ti Rotor Swinging Bucket</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Beckman L-80 Floor Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thickwall Polycarbonate Tubes (3.5mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TLA-100.3 Fixed Angle Rotor</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349481</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:261px;">Beckman TL-100 Tabletop Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

神经元亚细胞分离技术，允许被贩卖，并从突触蛋白的定量。如在1960年代末期最初所述，突触质膜相关蛋白，可以通过超速离心法在蔗糖密度梯度分离。一旦突触膜分离，大分子复合物被称为突触后密度可以随后，由于其不溶于洗涤剂中分离。用于分离突触细胞膜和突触后密度蛋白的技术基本上保持40年后相同的，并且被广泛应用于目前神经科学研究。本文将详细介绍与突触细胞膜及突触后密度采用不连续蔗糖密度梯度相关的蛋白质的分离。导致蛋白制剂适用于免疫印迹或2D DIGE分析。

在蔗糖密度梯度的制备方法应导致蔗糖（0.8，1.0和1.2M的蔗糖）的3摩尔的解决方案的明确分离。参见图3A为渐变的示例的蛋白质样品加入之前。如果梯度制备太多提前，或者如果它被制备的​​长凳上表面与来自其它设备的振动，梯度将受到损害，并适当分离，将无法实现。如果三种解决方案的明确分离是不可见的，最好是加入的蛋白样品之前，使一个新的梯度。参见图3B...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

本文将详细介绍与突触质膜上超速不连续蔗糖梯度相关蛋白的富集。随后的制备突触后密度蛋白也被描述。蛋白制剂适用于免疫印迹或2D DIGE分析。

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Research

JoVE Journal

Neuroscience

30.9K 次观看.  University of Toronto. 本文将详细介绍与突触质膜上超速不连续蔗糖梯度相关蛋白的富集。随后的制备突触后密度蛋白也被描述。蛋白制剂适用于免疫印迹或2D DIGE分析。 