We thank S. Raychaudhuri for providing the destabilized firefly luciferase mutant plasmid, and A. Glavis-bloom and N. Charan for technical assistance. This work was supported, in part, by grants from NIH (CA088868, GM060911, and CA182675).

1.试剂的制备<ol><li>制备细胞裂解缓冲液（50mM的Tris，pH值8.8，100mM NaCl的，5mM的MgCl 2的，0.5％的NP-40）。补充2毫摩尔DTT，1×完全蛋白酶的鸡尾酒，和250国际单位/毫升BENZONASE使用前。 </li><li>制备粒料缓冲液（20mM的Tris，pH值8.0，15mM的MgCl 2的）。补充2毫摩尔DTT，使用前1个完整的蛋白酶的鸡尾酒和250国际单位/毫升BENZONASE。 </li><li>制备3倍沸腾缓冲液（6％SDS，20毫摩尔Tris，pH8.0）中。使用前补充150毫摩尔DTT。 </li><li>准备使用荧光酶标仪检测读卡器低荧光DMEM培养基。混合的25mM葡萄糖，0.4mM的甘氨酸，0.4毫精氨酸，0.2mM的半胱氨酸，4.0 2mM谷氨酰胺，0.2mM的组氨酸，0.8毫异亮氨酸，0.8毫亮氨酸，0.8毫赖氨酸，0.2mM的甲硫氨酸，0.4mM的苯丙氨酸，0.4mM的丝氨酸，0.8毫苏氨酸，0.078毫色氨酸，0.4mM的酪氨酸，0.8毫缬氨酸，1.8毫氯化钙 ，0.81毫硫酸镁 ，5.33毫米氯化钾，44.0毫碳酸氢钠 ，110 mM氯化钠，0.9毫加入NaH 2 PO 4。调整的使用盐酸或NaOH至pH 7.4溶液的pH值。消毒通过过滤介质。 注意:此培养基含有除了铁具有高葡萄糖的标准DMEM培养基组分（NO 3）3，维生素和酚红被省略。酚红，核黄素和吡哆醛在定期的DMEM培养基与检测荧光信号21的显著干扰。如果没有这些成分的培养基是成功的活细胞成像GFP至关重要。当冷藏保存介质是12个月是稳定的。 </li></ol> Atxn1 82Q GFP的2降解测定<ol><li>板大约3×10 5个 HeLa细胞与DMEM介质35 MM平板补充有10％胎牛血清（FBS），使得后O / N培养细胞生长至40-60％汇合在转染时。 注意:需要板的数量是基于时间点和治疗D中的数下面旁切。 </li><li>转染的HeLa细胞与Atxn1 82Q-GFP / pRK5质粒0.3微克到根据制造商的指令9每孔用转染试剂。使含有DNA和转染试剂主染混合物和分装它每口井。 </li><li> 4-5小时后转染，检查GFP表达倒置荧光显微镜激发波长450-490纳米下的活细胞。返回细胞回孵化后成像。 注意:此时请遵守在核两种扩散GFP信号和GFP信号的小斑点。 </li><li>通过真空抽吸除去培养基，加入2毫升的新鲜的DMEM含有50微克/毫升CHX。处理细胞用于与收获前CHX 0，4，8，第12和16小时。为了检验蛋白酶体降解，包括在16小时作为对照处理的一个板蛋白酶体抑制剂MG132（10μM）。 </li><li>在每个时间点收获细胞的一个极板。通过负压吸引的移除介质ð用3毫升冰冷的1X磷酸盐缓冲盐水（PBS）洗涤板两次。管理单元冻结干冰板。 </li><li>最后时间点（16小时）后，刮去冷冻细胞（来自所有时间点）到150微升冰冷的细胞裂解缓冲液，并在冰上温育30分钟。 </li><li>离心在台式离心机的细胞裂解物在17000×g离心在4℃下15分钟。 </li><li>将上清转移，其中包含NP-40的可溶性（NS）蛋白，使用移液管的另一个管。 注:使用为上清液如果需要通过Bradford法测定蛋白质的浓度。 </li><li>轻轻加入约200μl的1×PBS中的管而不会干扰粒料冲洗粒料。小心地负压吸引或吸管除去PBS。重悬它们在150微升冰冷的细胞沉淀缓冲液中，接着15-30分钟温育在冰上。 注:重悬沉淀馏分含有NP-40的不溶物（NI）的蛋白质。参见图1B的诊断洗涤剂分馏的RAM。 </li><li>添加75微升3倍沸腾缓冲器的成NS个级分和NP-40的不溶物（NI）的级分从粒料再悬浮。加热该样品在95℃，5分钟的加热块上。 注:竹丛中的NP-40不溶性馏分加热和样品将明朗化后解散。 </li><li>添加SDS-凝胶上样缓冲液至煮沸的NS和NI的等分试样。加载来自所有时间点到SDS-PAGE凝胶收集的样品的等体积。从样品的体积装载对应于大约20微克的NS中的时间0。注收集:NS，以及SDS可溶性（SS）蛋白来自NI派，可以通过SDS分离胶来解决。与此相反，来自NI馏分SDS抗性（SR）聚集体卡在凝胶上样孔中（ 图1B）。为了提高免疫印迹检测，NI的双卷可装载。 </li><li>通过使用抗GFP抗体和增强化学发光（ECL）免疫印迹检测NS和SS Atxn1 82Q-GFP。 注意:使用此协议时，Atxn1 82Q在SS分数一般不太相比NS分数。是为了得到理想的信号再ECL曝光。 </li><li>从通过使用斑点印迹装置（ 图1B）滤波器相位差测定沉淀馏分审查SR Atxn1 82Q。简单地说，设立了点印迹装置拿着0.2微米醋酸纤维素膜。负载80-120微升煮沸的NI的入点印迹装置的各孔中。 注:由于在细胞中仅形成的SR聚集体的少量的，为对斑点印迹测定中，加载的SR分数是用于Western印迹分析的NS馏分的体积的约10-15倍的体积。 <ol><li>通过真空膜过滤样品后，停留在膜Atxn1 82Q-GFP聚集体可通过抗GFP免疫印迹9,12,22来检测。 注:请参阅之前的报道9,12,22过滤器阻滞实验的详细描述。此步骤是可选因为相对于按照本协议描述的短期转SS和NS分数SR Atxn1 82Q是最小的。此外，SR Atxn1 82Q的电平在很大程度上仍超过CHX追逐（ 图2B）相同。然而，它仍然是至关重要的，审查的SS Atxn1 82Q的量是否会受到影响随着时间的推移对在最初的实验中任何药物治疗或基因表达。 SS蛋白物种留在SDS-PAGE凝胶上样孔中可以通过免疫印迹进行检测。然而，该方法是不太敏感，并产生更多不同的结果（ 图1B）。 </li></ol></li></ol> NLS-荧光素酶-GFP的使用SDS-PAGE和Western Blot 3.降解测定<ol><li>板大约3×10 5个 HeLa细胞与DMEM介质35 MM平板补充有10％胎牛血清（FBS）。 O / N培养后，在转染时达到40-60％的汇合。所需板的数量是基于叔数IME点和治疗如下所述。 </li><li>转染的HeLa细胞用1.0微克NLS萤光素酶-GFP / pRK5质粒导入根据制造商的指令9每孔用转染试剂。使包含每个等分DNA和转染试剂以及一个主染混合物。 </li><li> O / N转（小时约15）后，检查GFP表达倒置荧光显微镜激发波长450-490纳米下的活细胞。返回细胞回孵化后成像。 注:扩散核GFP信号所用的大部分细胞（70％）的被观察到。细胞的一小部分（5％）有核聚集。 </li><li>通过真空抽吸除去培养基，加入2毫升的新鲜的DMEM含有50微克/毫升CHX。处理细胞与收获前CHX 0，1.5,3，4.5和6小时。为了检验蛋白酶体降解，包括在6小时作为对照处理的一个板附加蛋白酶体抑制剂MG132（10μM）。 </li><li>在每个时间点收获细胞的一个极板。除去由真空抽吸培养基并用3ml冰冷的磷酸盐缓冲盐水（PBS）洗涤板两次。管理单元冻结干冰板。 </li><li>最后时间点（6小时）结束后，细胞冷冻于所有时间点都刮入150微升冰冷的细胞裂解缓冲液，并在冰上温育30分钟。 </li><li>加SDS凝胶加样缓冲液以全细胞裂解物的2％的SDS的终浓度和50mM DTT。孵育一个热块上的样品在95℃下5分钟。 </li><li>通过SDS-PAGE和使用抗GFP抗体免疫印迹分析的NLS萤光素酶-GFP。加载来自所有时间点到SDS-PAGE凝胶收集的样品的等体积。卷装相当于大约20微克从样本在时间0收集全细胞裂解物的。 </li></ol>使用荧光酶标仪NLS-荧光素酶-GFP 4.实时降解测定<ol><li>种子约1×10 4个HeLa细胞与透明底的黑色96-孔组织培养板中。 O / N培养后，在转染时达到50-70％汇合。 注:60微升含有完全悬浮HeLa细胞培养基的直接接种到各孔中。不要播种后添加额外的媒体或岩石板上来回，否则电池可能会分布不均。 </li><li>转染的HeLa细胞与NLS-萤光素酶-GFP / pRK5质粒9的0.05-0.1微克到根据制造商的指示向各孔使用转染试剂。使含有DNA和转染试剂主染混合物和分装它每口井。三个井用细胞不与DNA，作为为每个处理条件背景荧光信号控制转染。 注:另一种方式，以减少染变化是播种之前转染细胞。然而，转染错折叠蛋白的细胞的大部分小号不重视或使用此方法显示减少生存能力。它很可能是一些细胞可能经不起表达毒性错误折叠的蛋白质时所造成的胰蛋白酶消化的应力。其结果是，总的荧光信号显著降低。 </li><li>转染后20-24小时，检查GFP表达倒置荧光显微镜激发波长450-490纳米下的活细胞。 </li><li>删除由负压吸引的网上平台。添加约200μl的1×PBS中，以每个孔，然后吸出它来除去DMEM培养基的剩余量。 </li><li>添加60微升低荧光DMEM培养基有5％FBS和50μg/ ml的放线菌酮。为了检验蛋白酶体降解，包括在一组样品额外蛋白酶体抑制剂MG132（10μM）。集井的一式三份的每个处理/条件。 注:MG132治疗被包括作为蛋白酶体降解的控制，因为它是非常重要的排除可能会导致FLUO的压降等因素rescence信号，包括细胞死亡和荧光淬灭。 </li><li>立即测量加入CHX后，荧光酶标仪GFP的荧光信号。 注:软件测量设置如表1所示。 </li><li>读板每隔一小时可达8-10小时。看完后，退回板回细胞培养孵化器。 </li><li>将数据导出为电子表格文件。使用平均值为每个多读取以及荧光强度。正常化每个数据点的值，以时间0的在各组的平均值。积归一化的荧光强度随着时间的推移， 如图4A和4B，以及图5，右面板。 </li><li>执行使用两因素ANOVA重复测量23两个条件之间降解率的统计分析。 注:在这种分析中，"荧光强度"是，而"treatm因变量耳鼻喉科条件"和"时间"是两个因素，而是在单独的时间点进行比较的数据，双向ANOVA重复测量分析在整个时间过程的两个治疗组之间的差异。 </li></ol>

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A., Wang, G., Nukina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+proteasomal+function+due+to+the+expression+of+polyglutamine-expanded+truncated+N-terminal+huntingtin+induces+apoptosis+by+caspase+activation+through+mitochondrial+cytochrome+c+release.">Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release.</a> Human molecular genetics. 10, 1049-1059 (2001).</li><li>Fribley, A., Zeng, Q., Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+inhibitor+PS-341+induces+apoptosis+through+induction+of+endoplasmic+reticulum+stress-reactive+oxygen+species+in+head+and+neck+squamous+cell+carcinoma+cells.">Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells.</a> Molecular and cellular biology. 24, 9695-9704 (2004).</li><li>Bence, N. F., Sampat, R. M., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+the+ubiquitin-proteasome+system+by+protein+aggregation.">Impairment of the ubiquitin-proteasome system by protein aggregation.</a> Science. 292, 1552-1555 (2001).</li><li>Chen, 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=Intrasarcoplasmic+amyloidosis+impairs+proteolytic+function+of+proteasomes+in+cardiomyocytes+by+compromising+substrate+uptake.">Intrasarcoplasmic amyloidosis impairs proteolytic function of proteasomes in cardiomyocytes by compromising substrate uptake.</a> Circulation research. 97, 1018-1026 (2005).</li><li>Link, C. 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=Conversion+of+green+fluorescent+protein+into+a+toxic,+aggregation-prone+protein+by+C-terminal+addition+of+a+short+peptide.">Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.</a> The Journal of biological chemistry. 281, 1808-1816 (2006).</li></ol>

The authors declare that they have no competing financial interests.

调节错误折叠的蛋白质的降解机制是用于维持细胞蛋白质的稳态必不可少的，并且它们可能代表用于治疗神经变性疾病和其他蛋白质错误折叠的疾病有价值的药物靶标。这里，检查错误折叠的蛋白质的降解测定法所述，用致病Atxn1蛋白（Atxn1 82Q）和核局部化的萤光素酶突变体（NLS-LucDM）作为例子。 为了检查降解Atxn1 82Q，具有半衰期超过18小时，我们介绍了细胞分离与经典CHX <su...

蛋白是在细胞中最丰富的大分子，和它们在几乎所有的生物过程中起重要作用。大多数蛋白的生物活性，需要其折叠成和维护，天然的三维结构。与异常的构象的蛋白不仅失去其正常功能，而且还常常形成削弱其它蛋白质的功能和对细胞有毒性，1,2-可溶性寡聚物种或聚集体。为了抵消蛋白质错误折叠，细胞同时采用分子伴侣，这有助于展开或部分折叠多肽达到自己的天然构象，和降解途径，从而消除错误折叠的蛋白质3。给出的复杂性和折叠处理的随机性质，蛋白错折叠是不可避免的，而且它可能无法在突变，生物合成的错误，以及翻译后的损害1的情况是相反的。因此，细胞最终依靠degradat离子通道以维持其蛋白质的质量。 细胞蛋白质的质量控制（PQC）系统的重要性，通过蛋白质错误折叠的疾病，包括癌症，糖尿病，和许多神经退行性疾病如阿尔茨海默氏病，帕金森氏病，肌萎缩性侧索硬化症，亨廷顿氏病（HD）的患病率强调，和小脑萎缩症（SCA）4,5。例如，在肿瘤抑制基因p53的突变是唯一最频繁的遗传病变肿瘤，具有〜所有病例6 50-70％相关联。 p53基因突变的相当大的部分是改变p53的构象的错义突变，导致聚集体7的形成。此外，扩大的polyQ绵延蛋白基因和病理与HD和SCA相关。当的polyQ伸展在受影响的蛋白质的长度超过一定THRES这些进步和常常是致命的疾病表现持有，并作为的polyQ拉伸的长度延长8变得越来越严重。 用于治疗这些疾病的有吸引力的方法是将治疗加强蜂窝PQC系统，特别是降解途径。然而，涉及有缺陷的蛋白质，特别是那些在哺乳动物细胞中的降解途径，仍然知之甚少。尽管已经认识到，蛋白酶体为错误折叠的蛋白质的降解非常重要，一个至关重要的问题仍然是未定义:蛋白质如何错误折叠被特异性识别和针对降解。此外，尽管PQC系统在细胞区室，包括细胞质中，内质网和线粒体已经确定，在细胞核中的PQC系统仍不清楚2。 由我们实验室最近的研究已经确定了在细胞核中识别并降低各种错折叠蛋白的一个系统哺乳动物细胞中9。这个系统由早幼粒细胞蛋白（PML），核蛋白质和三方含基序（TRIM）蛋白质家族的一员，并RNF4，含蛋白质的RING结构域的。 PML等几个TRIM蛋白，具有SUMO（小泛素样修饰符）E3连接酶的活性，有利于蛋白SUMO化10的特异性和效率。 RNF4属于一个小团体SUMO-针对性的泛素连接酶（STUbL），其中包含在除了能提供他们泛素连接酶的活性在11 RING结构域的一个或多个相扑相互作用基序（SIMS）的。我们发现，PML特异性识别通过，可以在错误折叠的蛋白质辨别特色鲜明离散底物识别位点错误折叠的蛋白质。结合后，PML标签错误折叠与SUMO2 / 3的聚链，两个几乎相同的哺乳动物SUMO蛋白可以形成由于内部SUMO化位点的存在聚链蛋白质。小号UMOylated错误折叠的蛋白质，然后通过RNF4，从而导致它们的泛素化和蛋白酶体降解的认可。我们进一步证明了PML-RNF4系统是针对神经变性保护重要的，因为缺乏在PML加剧SCA类型的小鼠模型1（SCA1）9的行为和神经病理学缺陷。 若要从可调节蛋白水平等细胞机制区分蛋白质降解，蛋白质周转率测量9。其中最常用的方法，以确定蛋白质周转是脉冲追踪和放线菌酮（CHX）追。这两种方法考察随着时间的推移，分别翻译，精通细胞和翻译，抑制细胞的总利息预先存在的蛋白质感兴趣的放射性同位素标记的蛋白质。但是，对于学习致病和错误折叠倾向的蛋白质的一个主要的挑战是，这些蛋白质的半衰期可以是非常罗NG。例如，共济失调蛋白1，共济失调蛋白7，亨廷顿，α突触核蛋白，和TDP-43都具有超过12的半衰期至24小时9,12-16。这些蛋白质的慢流动率排除使用CHX追分析的，因为携带这些蛋白质的细胞可能无法生存延长翻译抑制，特别是因为错误折叠的蛋白质本身可以是细胞毒性很强。用同位素标记的脉冲追踪分析还可以是对于那些高度聚集倾向的蛋白质具有挑战性。最脉冲追踪测定依赖于免疫沉淀到感兴趣的蛋白质从也放射性标记的所有其他蛋白质分离。此过程通常包括冗长免疫沉淀和洗涤过程，在此期间，SDS不溶聚集体可以形成，使得用SDS-PAGE电泳不精确的分析。 这里用一个缓慢的周转率说明9协议分析核错误折叠的蛋白质。包含的82个谷氨酰胺（Atxn1 82Q）拉伸共济失调蛋白-1（Atxn1）的致病形式用于此目的8。当在细胞中表达的Atxn1 82Q形式显微镜可见夹杂物的增强型绿色荧光蛋白（GFP）融合在细胞核中（ 图1A）。脉冲追踪分析表明Ataxn1 82Q的半衰期为18小时以上，9。 Atxn1 82Q是由具有不同特性的错折叠的构象，以及具有天然构象物种物种。它很可能是这些物种以不同的速率退化，并因此应该被单独分析。裂解物从Atxn1 82Q-GFP表达细胞被分馏成NP-40的水溶性（可溶于或生理盐水，可能代表天然蛋白质或错误折叠的单体/低聚物的蛋白）和NP-40的不溶性（NI，凝集/错折叠）部分。后者可进一步分为SDS可溶性（SS;可能无序集合体）或SDS抗性（SR;可能淀粉样蛋白原纤维）馏分（ 图1B）。 NS和SS馏分可以通过SDS-PAGE，随后通过Western印迹进行分析，而对SR分数可以通过过滤相位差测定，随后通过免疫印迹进行检测。 CHX追与洗涤剂分馏方法相结合，并且发现SS Atxn1 82Q的半衰期比NS Atxn1 82Q和总Atxn1 82Q（ 图2A）的短得多，这表明在SS馏分可以容易地识别和降解在细胞中9。因此，该方法提供了一个有力的工具来研究错误折叠的蛋白质的动态，并比较它们的降解模式。 我们还描述了适合于用于识别大分子或小分子化合物可以调节错误折叠的蛋白质的降解的高通量筛选的方法。此方法是基于萤火虫荧光素酶（LucDM）17，一个模型伴侣衬底的构象失稳突变体。我们有保险丝ðLucDM到一个核定位信号（NLS）来探测在此细胞区室的PQC系统，和GFP（NLS-LucDM-GFP）进行检测的便利性。 NLS-LucDM-GFP形式显微镜中的细胞（ 图3A）的一小部分可见核聚集体。类似Atxn1 82Q，NLS-LucDM-GFP-而不是它的野生型对应物NLS-LucWT-GFP-由SUMO2 / 3改性和调节由PML-RNF4通路9。 NLS-LucDM-GFP也形成SS和SR物种，虽然SS和SR物种的相对量相比NS是最小的，使用下文协议3所述的条件。为了简化分析，我们只分析SDS可溶性LucDM（包括NS和SS分数两者）通过SDS-PAGE和免疫印迹。重要的是，CHX追检测显示SDS可溶性NLS-LucDM-GFP的半衰期比其野生型对应物（ 图3B）的短得多，这表明LucDM为承认和降解系统中的特定基板错误折叠的蛋白质。 LucDM-GFP的降解导致整体荧光信号的显著下跌。因此，我们还开发了使用基于荧光微量测定细胞LucDM-GFP的实时检测的协议。许多高通量筛选（HTS）系统的药物或基因修饰的细胞聚集和细胞活力造成的异常蛋白质18-20发展。然而，很少HTSS是专门用于在哺乳动物细胞靶向降解而设计的。此协议可作为蛋白质表达，击倒，和药物治疗对细胞错折叠蛋白质降解的影响快速和大规模分析一个健壮的系统。使用HeLa细胞作为例子，下面我们描述了协议，这两个错误折叠的蛋白质的分析。该测定法还可以应用到其他的细胞系，虽然转染的条件和时间过程中可能需要对个别细胞系进行优化。

<table border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">11995-092</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Fetal Bovine Serum</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Lipofectamine 2000</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">NP-40</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">NP40S-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SDS</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">MG132</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8699</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Cycloheximide</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">C7698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Dithiothreitol (DTT)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:119px;">45000232</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Complete Protease Inhibitor Cocktail Tablets</td>
			<td style="width:175px;">Roche Boehringer Mannheim</td>
			<td style="width:119px;">4693159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Bio-Dot Apparatus&#160;</td>
			<td style="width:175px;">Bio-Rad</td>
			<td style="width:119px;">1706545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Living Colors GFP Monoclonal Antibody</td>
			<td style="width:175px;">Clonetech</td>
			<td style="width:119px;">632375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Anti-Actin mAb Rabbit, IgG</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">A45060-200UL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Amino acids</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td>Amino acids are used for making low fluorecence culturing medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Olympus IX-81&#160; Inverted Fluorescence Microscope</td>
			<td style="width:175px;">Olympus</td>
			<td style="width:119px;">IX71/IX81</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">96 Well Black TC Plate w/ Transluscent Clear Bottom</td>
			<td style="width:175px;">Sigma-Greiner</td>
			<td style="width:119px;">89135-048</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Fluorescence Bottom Plate Reader Infinite 200&#174; PRO</td>
			<td style="width:175px;">TECAN</td>
			<td style="width:119px;">Infinite 200&#174; PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Cellulose acetate membrane 0.2 &#181;m</td>
			<td style="width:175px;">Sterlitech</td>
			<td style="width:119px;">CA023001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Prism 5</td>
			<td style="width:175px;">GraphPad</td>
			<td style="width:119px;"></td>
			<td>Statistical analysis software</td>
		</tr>
	</tbody>
</table>

assays for the degradation of misfolded proteins in cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

在稳定状态下的分析，显微镜可见Atxn1 82Q-GFP核聚集体可以在30观察到- HeLa细胞的50％的转染（ 图1A）后20小时。使用抗GFP抗体的NS和SS级分的Western印迹分析显示100 kDa和150 kDa的标记之间Atxn1 82Q-GFP的一个独特的带，对应于该蛋白质的分子量（ 图1B）。 Atxn1 82Q-GFP在SR分数可以通过过滤相位差测定法，或通过SDS-PAGE和免疫印迹如在浓缩胶（ 图1B）</st...

Watch this Scientific Journal Video about Assays for the Degradation of Misfolded Proteins in Cells at JoVE.com

Assays for the Degradation of Misfolded Proteins in Cells

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

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Biology

11.9K 次观看.  University of Pennsylvania Perelman School of Medicine. This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening. 

视频: Assays for the Degradation of Misfolded Proteins in Cells

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

科研

生物学

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

重组β-catenin的降解<em&gt;爪蟾</em&gt;卵提取物

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

比较GTP酶结合蛋白的使用竞争性试验的亲和

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...