这项工作是由杜塞尔多夫大学医学系的Forschungskommission授出的的神经元ERANET发现（BMBF 01EW1003）CK和JPH，DFG（高1679/3-1; GRK1033）CKVB支持。

1。聚集小体细胞转染制备的单体红色荧光蛋白（MRFP）或增强型绿色荧光蛋白（EGFP）标记的人类全长（FL）DISC1 <ol><li>种子1.5×10 6/10厘米的菜人类神经母细胞瘤民族解放阵线（NLF费城儿童医院，费城，PA）在10 10 cm培养皿中的细胞和生长过夜。 NLF培养细胞在RPMI-1640培养基中补充有10％FBS，青霉素/ L-谷氨酰胺Strepomycin。第二天，细胞应在70％至80％汇合。 </li><li>瞬时转染每个10 cm培养皿中，用15微克质粒DNA和Metafectene的转染试剂（Biontex，Martinsried，德国）。简言之，为10 cm培养皿中，加入15微克质粒DNA和30μlMetafectene转染到750微升的Opti-MEM，混合，并在室温孵育20分钟。 5菜肴，与pcDNA3.1 MRFP /绿色荧光蛋白标记的人（FL）的DISC1和5菜的绿色荧光蛋白的质粒单独转染。 </li></ol> 2。聚集小从转染供体细胞的分离纯化这里所描述的协议是从脑组织13和协议来隔离大团聚体和aggresomes 14的一个方法来净化路易机构的组合。由于现有的方法是基于使用的洗涤剂，洗涤剂 - 自由蛋白细胞入侵性的测定中的应用程序的隔离是至关重要的。 <ol><li>转染后48小时，监测表达eGFP和mRFP/eGFP-DISC1，在荧光显微镜下（ 图1），确认形成aggresomes。 </li><li>刮pH为7.4，用400μl的PBS中每个用PBS液洗涤细胞，添加1X蛋白酶抑制剂鸡尾酒（Roche公司，印第安纳波利斯，IN）和使用一个Precellys24均化器（贝尔坦技术，法国）破坏细胞。简单地说，加1 ml细胞悬浮液2毫升的裂解管，并添加10个陶瓷珠。裂解细胞，用3×60秒的脉冲。的过程中的机械力可能增加样本内的温度超过37°C，因此冰后的裂解过程中，应采取放松的样品。 </li><li>细胞在冰上冷却，加10毫米的MgCl 2，40 U / ml的DNA酶A和孵育60分钟，在37°C </li><li>准备解决方案，为80％，50％，20％的蔗糖（重量/体积），在PBS pH为7.4（10毫升）。 </li><li>准备15毫升falcon试管中开始，用2毫升80％的蔗糖，其次是50％和20％的蔗糖梯度。慢慢倒入梯度，要小心，不要去打扰已经浇层。层之上的梯度和离心机消化的溶胞产物在4℃下15分钟，在1000×g离心</li><li>仔细收集相间50-80％蔗糖层之间，用移液管和混合用5倍体积的PBS pH值7.4。在4℃下离心15分钟，在1000×g离心重复洗2次。在此相间，aggresomes的荧光，在显微镜下可以可视化的UV光。 </li><li>弃去上清并悬浮颗粒在250-500微升PBS pH值7.4。该颗粒包含的：纯化aggresomes（ 图2）。 </li></ol> 3。收件人的SH-SY5Y细胞治疗mRFP/eGFP-DISC1 Aggresomes <ol><li>种子5×10 4个收件人的SH-SY5Y人成神经细胞瘤细 ​​胞组成型表达的GFP-DISC1（598-854），在24孔板的各孔中的无菌玻璃盖玻片上。 SH-SY5Y细胞培养在DMEM/F-12培养基与青霉素/链霉素，非必需氨基酸（NEAA）和10％FBS。 </li><li> 24小时后，每孔加入10μl，孵育48-72小时。 </li><li>洗涤细胞3×，pH为7.4的PBS和急冷5分钟，用0.​​04％的台盼蓝的细胞外荧光。 </li><li>在冰上10分钟，在PBS pH值7.4用4％PFA固定细胞，用无菌H 2 O洗一次并挂载在载玻片上的盖玻片的ProLong用DAPI安装培养基（Invitrogen公司，美国）的黄金。 </li><li>确认的吸收/入侵外源性应用aggresomes的共存与招募栈Z-图像所表达的受体细胞的蛋白质。 </li></ol>然而，外源蛋白的吸收/入侵可能没有本质的招聘宿主细胞蛋白的同时检测。在我们的例子中MRFP标签DISC1 aggresomes招可溶性GFP标记DISC1（598-854）由宿主细胞表达的蛋白质（参见图3）。图片采取了蔡司LSM 510共聚焦或一个蔡司Axiovision Apotome2显微镜。 4。产生的重组DISC1（598-785） 在以前的出版物中，我们分析自基于自缔合结构域的存在下相互作用的能力的各种DISC1蛋白片段。其中所有的蛋白质片段测试人类DISC1（598-785）显示最强的多聚倾向4（ 图4）。因此，人类DISC1（598-785）被克隆到pET15b（否沃根，麦迪逊，威斯康星州）的N-末端含有6 -组氨酸标记，在大肠杆菌中表达大肠杆菌 BL21（lDE3）罗塞塔（Novagen公司，美国），在变性条件下在8摩尔/升尿素所描述4纯化。总之，该协议被概述如下。 <ol><li> BL21（IDE3）罗塞塔E.大肠杆菌生长在含有5mM的-精氨酸盐酸，5mM的MgSO 4干燥，100微克/毫升carbencillin，35微克/毫升氯霉素的OD 600的 2×500毫升2YT：0.6-0.8和DISC1（598-785）表达诱导用1mM IPTG。 </li><li> DISC1（598-785）表达在37℃下4小时，通过离心收获细菌表达终止。 </li><li>将细菌沉淀悬浮于50ml TE缓冲液（50mM的pH为8.0的Tris-HCl，5mM EDTA）中加2mM的PMSF，1％TX-100，250微克/毫升溶菌酶的20mM MgCl 2和400 U/50毫升DNA酶一若要确保完全裂解，反应在室温缓慢搅拌下温育30分钟。 </li><li>添加10毫米β-mercapt“oethanol（ME）和500 mM NaCl和另一个30分钟的孵育。 </li><li>自旋向下的细菌包涵体20.000克30分钟，在4°C。 </li><li>重悬沉淀在50ml提取缓冲液中含有50mM的Tris pH值8.0，5mM咪唑，500mM NaCl的，​​8M尿素和10mM的β-ME和删除在20.000克碎片通过离心30分钟，在4℃下</li><li>用提取缓冲液，洗净的Ni-NTA琼脂糖矩阵。孵育再悬浮，precleared的的蛋白质提取物用Ni-NTA基质2小时，在RT。 </li><li>用提取缓冲液含有12 mM咪唑的Ni-NTA基质洗净。 </li><li>用15毫升的洗脱缓冲液含有300mM咪唑的50mM Tris，500mM NaCl的，​​8M尿素，1mM的PMSF，5mM EDTA和10mM的β-ME，慢慢洗脱蛋白质。 </li><li>透析蛋白逐步PBS pH7.4的含10mMβ-ME。 </li></ol>从1 L细菌开始培养它可以分离高达50毫克重组DISC1（598-785）PROTEIN。 α-突触核蛋白复性对于实验与α-突触核蛋白，500微克的重组蛋白（Sigma-Aldrich公司，美国）溶解在PBS中的浓度为1毫克/毫升。为了获得低聚物，重折叠进行过夜，在37℃下，如先前的出版物10中所述。 5。标签重组DISC1（598-785）DyLight594的<ol><li>之前在随后的贴标过程中，DISC1（598-785）蛋白在被释放β-ME的。因此，该蛋白质是透析3次，PBS pH7.4的1:2,000稀释。 </li><li>标签DISC1 1毫克（598-785）马来酰亚胺（DyLight 594的Thermo Scientific，USA）根据制造商的说明。总之，1毫克/毫升的蛋白质在P​​BS pH7.4的溶解，和添加5mM的TCEP复原游离的巯基。加入20μl的DMF中溶解的染料的反应，并在室温孵育2小时。 </li><li>透析蛋白3×PBS pH值7.4在1:2000的比例为2小时，每个。 </li></ol>为了进一步增加标记的蛋白质的纯度的His 6标签的基础上的Ni-NTA柱进行亲和纯化。 <ol start="4"><li>洗净1毫升Ni-NTA的基质（Qiagen，德国）用10ml PBS pH为7.4。 </li><li>标记，透析蛋白的列，让它慢慢运行在列。 </li><li>洗净，用3×20毫升PBS的pH为7.4的蛋白质</li><li>同时监测的着色的频带上的Ni-NTA柱减少的洗脱液体积，pH为7.4，500mM咪唑滴加用PBS洗脱该蛋白质。 </li><li>透析的洗脱液3×10mM的NAPI pH为​​7.4的稀释度1:2000每次2小时。 </li><li>使用无菌的0.45μm的注射器过滤器进行过滤的洗脱液，在5×100微升样品在液氮中急速冷冻的等分试样。蛋白质的总量应为0.5 - 1微克/毫升在每100μl等分。 </li></ol> 可选的concentrATION步骤<ol start="10"><li>对于大鼠注射stereotactical，蛋白质浓缩10倍速度真空（Eppendorf公司选矿厂5301）。最后的缓冲条件为100毫米NAPI。 </li></ol> α-突触核蛋白的标签的标记和纯化的重组α-突触核蛋白（Ni-NTA柱）是并行执行的DISC1（598-785）。总的起始原料为500微克，而不是1毫克DISC1（598-785）。 6。收件人的SH-SY5Y细胞的标记重组DISC1（598-785）蛋白的治疗<ol><li>种子5×10 4收件人的SH-SY5Y人成神经细胞瘤细 ​​胞组成型表达的GFP-DISC1（598-854），在24孔板的各孔中的无菌玻璃盖玻片上。 </li><li>添加标记的蛋白质为5-10微克/毫升的浓度在细胞培养基中孵育48-72小时。 </li><li>洗涤细胞3×，pH为7.4的PBS和淬火提取用0.04％的台盼蓝5分钟ellular荧光。 </li><li>在冰上10分钟，在PBS pH值7.4用4％PFA固定细胞，用无菌H 2 O洗一次并挂载在载玻片上的盖玻片的ProLong用DAPI安装培养基（Invitrogen公司，美国）的黄金。 </li><li>确认的摄取外源重组蛋白的共存与招募蛋白表达的受体细胞所产生的激光扫描共聚焦显微镜图像栈Z-/入侵。然而， - 招聘内源性蛋白质可能基本上不被检测到入侵宿主细胞蛋白质平行，Z堆叠图像仍可以确认的侵入性的蛋白质的性质。 </li></ol>在我们的例子中录制。 DISC1（598-785）*至少部分新兵可溶性GFP标记DISC1（598-854）由宿主细胞表达的蛋白质（参见图5A）。对于重组α-突触核蛋白入侵但没有招聘所示（ 图5B）。照片拍摄在蔡司LSM 510共聚焦一个蔡司Axiovision Apotome2显微镜。 该注射液的浓缩（约4微升2.5微克/微升），内侧前额叶皮质的查询结果在注射部位周围的蛋白质可被检测即使灌注后的动物传播到标记为DISC1（598-785）*在PBS pH7.4的4％PFA诺丹明filterset。 DISC1（598-785）的是，在我们的例子中采取了由不同的神经元数目，并可以监控与Z堆叠成像（ 图6）。 在一般情况下，注射其他的蛋白质这里所使用的那些不必然导致细胞的入侵。入侵事件本质上是依赖于该蛋白质的性质，但一个干净的净化是一个先决条件，作进一步的分析。 7。代表性的成果从NLF细胞组成的重组FL DISC1-EGFP纯化Aggresomes入侵收件人SH-SY5Y CE的LLS效率低（约0.3％5），如看到的用共聚焦显微镜（ 图3）的共定位。据此前报道，DISC1（598-785） 从 E的表达和纯化大肠杆菌形成多聚体4也同样侵入性细胞的效率为约20％5（ 图5A）作为阳性对照（ 图5B），它被应用于平行类似的α-共核蛋白。从E.标记的重组DISC1（598-785）表达和纯化大肠杆菌细胞侵入神经 ​​元在体内的多聚蛋白立体定向注射到老鼠（ 图6 PUM，贝德，休斯顿，科思，未发表）内侧前额叶皮质的。 <img src="/files/ftp_upload/4132/4132fig1.jpg" alt="图1" /> 图1。NLF瞬时表达的神经母细胞瘤细 ​​胞：FP-DISC1（红色）。红色荧光aggresomes的可DETE反恐执行局在细胞内。 <img src="/files/ftp_upload/4132/4132fig2.jpg" alt="图2" /> 图2纯化的MRFP标签后DISC1 aggresomes，在蔗糖密度梯度纯化。 <img src="/files/ftp_upload/4132/4132fig3.jpg" alt="图3" /> 图。荧光图像的入侵aggresomes。 ，进行纯化，MRFP标签flDISC1 aggresomes和培养SH-SY5Y神经母细胞瘤细胞过表达可溶性GFP-的标签DISC1（598-854）。吸收标记aggresomes的监测上的激光扫描显微镜（蔡司LSM 510）Z-叠加成像。 <img src="/files/ftp_upload/4132/4132fig4.jpg" alt="图4" /> 图4。SEC高调的建议。 DISC1（598-785）含有S704和C704的单核苷酸多态性（SNP）。两个变种中断，有序的寡聚化和形成的超高分子量多个imers（红色箭头）。此图像被修改从出版物Leliveld 等人 2009年，生物化学。 <img src="/files/ftp_upload/4132/4132fig5.jpg" alt="图5" /> 图5。荧光Z堆栈的图片侵入性的DyLight的标记重组DISC1（598-785）。 SH-SY5Y神经母细胞瘤细胞表达可溶性的GFP-DISC1（598-854）的孵育与标记的重组蛋白在5微克/毫升的浓度。 （A）红聚集入侵的建议，。 DISC1蛋白（598-785）和黄点表示聚集的招聘可溶性GFP-DISC1（598-854）。 （B）重组α-突触核蛋白（红色）侵入细胞没有招聘的频率在20％左右。 <a href="/files/ftp_upload/4132/4132fig5large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/4132/4132fig6.jpg" alt="图6" /> 图6。免疫荧光图片注入的标记，重组DISC1（598-785）蛋白。 Z堆叠成像确认记录的存在下。 DISC1蛋白（598-785）的大鼠皮层神经元与神经元的的核（NEUN，绿色）的抗体染色。

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Y. <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+aggresomes+and+other+large+aggregates.">Isolation of aggresomes and other large aggregates.</a> Curr. Protoc. Cell Biol. Chapter 3, 1-9 (2010).</li><li>Korth, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DISCopathies:+brain+disorders+related+to+dysfunctional+DISC1.">DISCopathies: brain disorders related to dysfunctional DISC1.</a> Rev. Neurosci. 20, 321-330 (2009).</li></ol>

没有利益冲突的声明。

在这项研究中，我们描述的净化起的mRFP/eGFP-DISC1 aggresomes转染神经母细胞瘤细 ​​胞系，准备和标签的重组DISC1蛋白种类和他们的应用程序在入侵细胞的受体细胞在体外和体内实验。 从协议开发的纯化的固有mRFP/eGFP-DISC1 aggresomes隔离Lewy体样的结构和较大的聚集体13，14，但修改，以避免使用洗涤剂，以尽量减少可能发生的假阳性细胞入侵的风险由于洗涤剂促进的膜渗透。 一个可能的未来的改善可能是在进一步提高纯度的aggresomes通过超声完全分开，其余的细胞膜和细胞骨架的aggresomes。通过提高整体纯度总的入侵事件，，马大aggresomes记录可以增加5。有限的定义，我们建议的3相蔗糖是否是足够aggresomes其他蛋白物种，以进行测试，在这个意义上的协议这里列出应被视为单个优化的一个起点。的的纯化aggresomes被证明是相当强大的，在我们的手中，不用洗衣粉的洗涤步骤（），以消除痕迹蔗糖的整体收益率并没有显着减少。 在以前的出版物中，我们描述了低聚物的DISC1组装是依赖于不同的多聚化域的C-末端4（ 图4）中。我们选择了这个片段在大肠杆菌中的可溶性表达的重组大肠杆菌和随后的Ni-NTA净化。为了监视它的多聚化，并比较其与所述的细胞侵入性的蛋白，如α-突触核蛋白细胞的侵袭性，我们标记DISC1（598-785）与荧光染料DyLight594中，和比较其同样标记的α-突触核蛋白细胞的侵袭。重组DISC1片段细胞侵袭力急剧增加相比，本地的，全长DISC1 aggresomes，可能是由于其更小的尺寸和更高的纯度。同样，的纯度似乎发挥了决定性的作用，在细胞侵袭。 在我们的例子中的侵入aggresomes招募可溶性同源蛋白的重组表达的靶细胞线在一种可溶性的， 即细胞分散的形式。一种荧光蛋白（ 如 GFP或RFP）中的表达，在受体细胞线是一个优点，因为它允许通过Z堆叠成像侵入aggresomes和重组蛋白的共定位在受体细胞的限制之内。 从大肠杆菌表达和纯化的细胞侵入性的DISC1 aggresomes和多聚体片段大肠杆菌可能是一个定义功能的蛋白质构象病有关DISC1，DISC1opathies 15。 故障排除： 聚集小产量低蔗糖密度梯度纯化后： 在该协议中的蔗糖密度梯度介绍以及与eGFP/mRFP-DISC1（FL）aggresomes的。其他蛋白质组成的aggresomes可能会对因此可变尺寸应该优化的蔗糖浓度，由其他用户。 纯化的重组蛋白不足： 也将被标记的重组蛋白在纯化过程中的任何细菌污染物在后面的步骤的协议。为了避免污染，蛋白的SDS-PAGE上运行，并确认该重组蛋白是至少95％纯的，通过用考马斯亮蓝染色。为了保证最高的质量和产量，为每个蛋白质的协议进行优化。 低标签效应FICIENCY的重组蛋白： 包含游离SH基团的任何污染，会降低有效标记的蛋白质。因此，广泛的透析和Ni-NTA柱纯化​​是强制性的。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen公司</td><td> 11875-093 </td><td>介质是依赖于宿主细胞系。应当由用户确定的最佳的受体细胞线。 </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen公司</td><td> 11320-033 </td><td>介质是依赖于受体细胞线。应当由用户确定的最佳的受体细胞线。 </td></tr><tr><td> PBS </td><td> Invitrogen公司</td><td> 14190-250 </td><td></td></tr><tr><td> Metafectene </td><td> Biontex </td><td> T020-1.0 </td><td>其他转染试剂可以工作的很好，但我们的协议没有测试过。 </td></tr><tr><td> Ni-NTA琼脂糖</td><td> Qiagen公司</td><td> 30210 &lt;/ TD&gt; <td>自Qiagen Ni_NTA琼脂糖做实验，其他供应商应正常工作。 </td></tr><tr><td> DNA酶I </td><td>罗氏公司</td><td> 04716728001 </td><td>有没有必要为无RNA酶的DNA酶的过程中aggresomes纯化。 </td></tr><tr><td>蔗糖</td><td> Sigma-Aldrich公司</td><td> S0389 </td><td></td></tr><tr><td>的Opti-MEM </td><td> Invitrogen公司</td><td> 31985-062 </td><td>无血清培养基工程，以及</td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15140122 </td><td>补充SH-SY5Y和NLF中等</td></tr><tr><td>非必需氨基酸（NEAA） </td><td> Sigma-Aldrich公司</td><td> M7145 </td><td>补充SH-SY5Y中</td></tr><tr><td>台盼蓝0.4％ </td><td> Sigma-Aldrich公司</td><td> T8154 </td><td>有毒试剂</td></tr><tr><td> DyLight 594马leimide </td><td>赛默飞世尔科技</td><td> 46608 </td><td>减少半胱氨酸的反应性染料，以形成稳定的硫醚键。也可在其他颜色。 </td></tr><tr><td>延长黄金与DAPI </td><td> Invitrogen公司</td><td> P36935 </td><td>这种抗淬灭液体封固了骄人的业绩，在我们的手中。 </td></tr><tr><td>蛋白酶抑制剂鸡尾酒</td><td>罗氏公司</td><td> 11873580001 </td><td> 1台溶解在500μlH 2 O为100X解决方案。 </td></tr><tr><td>合成的α-突触核蛋白</td><td>西格玛</td><td> S7820 </td><td>再折叠如文本中所述。 </td></tr><tr><td></td><td></td><td></td><td> 表的特定试剂。 </td></tr><tr><td> Precellys 24 </td><td>贝尔坦技术</td><td> 03119.200.RD000 </td><td>我们没有测试过其他机械均化比这其他nizers。其他洗涤​​剂均匀化方法可能会奏效，但此协议还没有经过测试。 </td></tr><tr><td> 5301集中器（Speed​​Vac离心） </td><td> Eppendorf公司</td><td> 5301 000.210 </td><td>只有必要的，如果蛋白质以集中。 </td></tr><tr><td> LSM 510和Axiovision Apotome2的</td><td>蔡司</td><td>制造商目录</td><td>这两种显微镜包庇的执行栈Z-成像能力。这是一个先决条件，固体和认真评估的入侵事件。 </td></tr><tr><td>细胞培养塑料材料</td><td> Nunc公司</td><td> 10 cm培养皿24孔板＃142475＃172958， </td><td>不同的塑料材料的使用可能会影响重组的蛋白质或aggresomes和塑料表面的相互作用。我们还没有对这里所描述的那些以外的材料进行测试。 </td></tr><tr><td></td><td></td><td></td><td> 表的具体的材料和设备。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 数 </td><td> 缓冲区的名字 </td><td> 内容 </td><td> 评论 </td></tr><tr><td> 1。 </td><td>细菌的生长介质</td><td> 16克/升细菌用胰蛋白胨，10克/升的Bacto酵母提取物，5克/升的NaCl，5mM的-精氨酸盐酸盐，5mM的MgSO 4干燥，100微克/毫升carbencillin的，35微克/毫升氯霉素</td><td>细菌生长到OD 600：0.6-0.8表达在1 mM IPTG。 </td></tr><tr><td> 2。 </td><td>细菌重悬缓冲液</td><td>的50mM的Tris-HCl pH为8.0，5mM的EDTA，2mM的PMSF，1％TX-100，250微克/毫升溶菌酶，20mM的MgCl 2和400 U/50毫升DNA酶I </td><td>从1 L Resupend细菌沉淀奥雅纳rnight文化重悬缓冲液50毫升。 </td></tr><tr><td> 3。 </td><td>蛋白质提取缓冲液</td><td>的50mM Tris pH为8.0，5mM咪唑，500mM NaCl的，​​8M尿素和10mM的β-ME </td><td></td></tr><tr><td> 4。 </td><td> Ni-NTA柱洗涤缓冲液</td><td>的50mM Tris pH为8.0，5mM咪唑，500mM NaCl的，​​8M尿素和10mM的β-ME，12 mM咪唑</td><td>修改提取缓冲液12 mM咪唑。 </td></tr><tr><td> 5。 </td><td> Ni-NTA柱洗脱缓冲液</td><td>的50mM Tris，500mM NaCl的，​​300 mM咪唑，8M尿素，1mM的PMSF，5mM EDTA和10mM的β-ME </td><td>的洗脱缓冲液含有10mMβ-ME，透析购买。 </td></tr><tr><td> 6。 </td><td> PBS </td><td> 8克/升的NaCl，0.2克/升的氯化钾，1.44克/升的Na 2 HPO 4，0.24克/升KH 2 PO 4，调整pH至7.4 </td><td></td></tr><tr><td>7。 </td><td>标记缓冲液</td><td> PBS含有5 mM TCEP </td><td></td></tr><tr><td colspan="4"> 配方表。 </td></tr></tbody></table></td></tr></tbody></table>

generation, purification, and characterization of cell-invasive disc1 protein species

蛋白质聚集的被看到作为一个一般标志的慢性，退行性脑条件类似，例如，在神经退行性疾病中阿尔茨海默氏病（Aβ，tau蛋白），帕金森氏病（α-突触核蛋白），亨廷顿氏疾病（多聚谷氨酰胺，亨廷顿），和其他。蛋白质聚集被认为是发生不安proteostasis， 即错误折叠的蛋白质的产生和降解之间的不平衡。值得注意的是，同样的蛋白质被发现聚集在散发性疾病是罕见的变异的突变体在家族形式。 精神分裂症是一种慢性进行性脑部疾病，在许多情况下，伴随着一个永久性的，不可逆的认知功能障碍。在候选基因的方法，我们调查是否破坏精神分裂症1（DISC1），一个基因在苏格兰的家庭与联动，慢性精神疾病1，2，可以发现，作为不溶性聚集在腹腔n的3精神分裂症的散发病例。使用SMRI CC，我们发现在约20％的病例与CMD，但不正常的控制或与神经退行性疾病的患者的肌氨酸不溶于DISC1免疫生化分离后。随后的研究表明， 在体外的聚集倾向的DISC1疾病相关的基因多态性S704C 4的影响，并且在体外产生DISC1 aggresomes细胞侵入性的5，类似于已Aβ6所示，tau蛋白7-9，α -突触核蛋白11，10，多聚谷氨酰胺或SOD1的聚集体12。这些发现提示我们建议用CMD的至少一个子集的情况下，那些凝集DISC1可能会对蛋白质构象疾病。 在这里，我们将介绍如何在哺乳动物细胞中产生DISC1 aggresomes，净化他们的蔗糖梯度，并使用它们细胞侵袭STUDI的ES。同样，我们将介绍如何生成一个专门的多聚C-端DISC1片段，标签和净化细胞侵袭的研究。使用重组的多DISC1我们实现类似的细胞侵袭类似的标记合成的α-突触核蛋白片段。我们还表明，该片段在体内时，立体定位注射到受体动物的大脑。

Watch this Scientific Journal Video about Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species at JoVE.com

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

的产生，纯化和细胞浸润的细胞，胞浆全长DISC1蛋白aggresomes从细胞培养和标记，多聚重组DISC1蛋白片段中<em&gt; E.大肠杆菌</em&gt;进行说明。为受体细胞在细胞培养和神经元细胞的侵袭<em在体内</em&gt;后脑接种stereotactical。

侵入细胞DISC1蛋白种类的产生，纯化及表征

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

generation-purification-characterization-cell-invasive-disc1-protein

Research

JoVE Journal

Biology

13.6K 次观看.  Medical School Düsseldorf, Germany. 的产生，纯化和细胞浸润的细胞，胞浆全长DISC1蛋白aggresomes从细胞培养和标记，多聚重组DISC1蛋白片段中 E.大肠杆菌进行说明。为受体细胞在细胞培养和神经元细胞的侵袭

视频: Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...