我们感谢李劳里的手稿的批判性阅读。三元催化器是由美国心脏协会博士前奖学金（12PRE6590007）的支持。 MRB是由美国国家癌症研究所的训练津贴（T32 CA119925）的支持。 SSH是由美国国立卫生研究院（R01DK078640）的支持。 EL是由美国国立卫生研究院（R01GM081635和R01GM103926）的支持。

非洲爪蟾卵提取物1。准备注：每个青蛙产生大约1毫升可用卵提取物。从10青蛙提取物通常被制备在同一时间，并如下所述缓冲器的容量是用于执行10蛙爪蟾卵提取物制备。缓冲体积可以相应于更大或更小卵提取物的制剂来调节。以这种方式产生的Preps始终得到蛋白质浓度≥50毫克/毫升。当由两个人进行收集鸡蛋，将它们加工成提取物的过程中是最有效的。 （对于基本的青蛙养殖技术，看西伯等。33）。 <ol><li>鸡蛋收集<ol><li>以黄金的青蛙，用孕马血清促性腺激素100单位（PMSG）从新鲜出炉的250 U / ml的股票每次注射雌蛙。使用3毫升结核菌素注射器用27号针头注入皮下，与该斜面针上，放入背淋巴囊，位于大约1厘米的距离沿青蛙的腿的长度的切口变色中线。 </li><li>在水店催芽青蛙（加20 mM氯化钠，见西伯等。33）在18°C为5-10天。对于站在水箱系统，动物密度约为4升每雌蛙水。注：需要吸生效的最短时间是5天，吸的影响后10天穿脱。 </li><li>准备0.5X Marc的修改铃声（MMR）从20倍MMR原液。 20倍的MMR由2M氯化钠，40mM的氯化钾，40mM的氯化钙，20mM的氯化镁，和100mM 4 - （2 - 羟乙基）-1 - 哌嗪乙磺酸（HEPES），pH为7.4。 </li><li>成立水桶的所有注入青蛙（每4升一桶1青蛙）。注：虽然不止一个青蛙可以放在同一个桶的鸡蛋收集，如果其中一只青蛙规定主要质量差的鸡蛋，努力很大一部份将被要求从那些适合制作提取物分离出质量低劣的鸡蛋。因此，最大化的青蛙在同一水箱的数量，以减少缓冲的用于蛋的量收集是不值得的风险。 </li><li>注入750ü人绒毛膜促性腺激素（HCG）到使用27号针头在1.1.1中描述的每个青蛙背淋巴囊。 </li><li>每个HCG注射青蛙放入含0.5倍MMR个人4升桶冷却到16℃。 </li><li>将容器在16℃培养箱中培养青蛙收集鸡蛋O / N（15-16小时）。注：保持适当的温度是整个过程的关键，包括从收集鸡蛋制备卵提取物。 </li></ol></li><li> Dejellying鸡蛋 注意：鸡蛋都覆盖着透明带，必须事先作出提取液中除去。卵的自发裂解的可能性增加作为betwee时间Ñ​​产蛋和提取准备增加。因此，为了继续通过以下步骤尽可能快是很重要的。 <ol><li>制备4升1X MMR的50毫升0.1×MMR的，和400ml的2％的半胱氨酸，pH值7.7，在蒸馏水中制成的。保持所有的解决方案在16°C。 </li><li>驱逐额外的鸡蛋，轻轻挤压青蛙的腰部和腹部。 </li><li>取出青蛙和大部分的孕产妇死亡率，离开鸡蛋每个桶中约1-200毫升的MMR。 </li><li>用移液管去除杂物，并评估卵子的质量：高质量的鸡蛋一般都标在深肤色动物半球和浅色的植物性半球之间的明确分离，具有最高的暗到光对比度。丢弃移液管出现拉丝，斑驳，或裂解（白色和浮肿），因为它们会降低提取物的整体素质任何鸡蛋。如果鸡蛋&gt; 10％质量较差，整个一批应该被丢弃。 </li><li>结合蛋放入500毫升的玻璃烧杯中，倒入尽可能多的MMR，同时尽可能保持鸡蛋淹没。 </li><li>通过轻轻旋转冲洗鸡蛋与MMR的蛋量的两倍。重复两次，并删除任何碎片或明显质量差的鸡蛋。 </li><li>加入大约100ml 2％半胱氨酸的玻璃烧杯中，轻轻摇动混合，并让鸡蛋定居5分钟，在16°C。倒掉半胱氨酸。注：Dejellying的标志是果冻外套浮上面的蛋和蛋的更紧凑的包装的逐步外观，因为它们现在占据更小的体积，而不果冻外套。 </li><li>加入另外100ml 2％半胱氨酸，轻轻地搅动，等待5分钟，然后慢慢倒出半胱氨酸。重复，直到鸡蛋已经成为紧密压实（通常由第三半胱氨酸治疗）。注意：如果鸡蛋留太长时间半胱氨酸，它们容易溶解。同样，dejellied鸡蛋是脆弱的，容易出现机械溶解，如果他们有传言太大力，或者如果他们暴露在空气中。一旦蛋已经dejellied，能够迅速进行离心分离的步骤是很重要的。 </li><li>倒出半胱氨酸，并冲洗掉果冻大衣等杂物轻轻洗蛋1X孕产妇死亡率。小心地倒出缓冲沿烧杯的一侧。重复两次或两次，直到MMR解决方案不再是阴天。而用1X孕产妇死亡率继续冲洗用移液管将坏鸡蛋。 </li><li>用30毫升0.1倍MMR进行最后的温柔冲洗，然后轻轻倒出尽可能多的缓冲区越好。再次，删除任何明显的坏蛋。 </li></ol></li><li>包装和鸡蛋破碎，离心 注意：下面描述的提取物被用于β-连环蛋白降解的抑制细胞生长因子提取物（中期II-被捕）的变体。相反，用于细胞周期的研究低速和高速提取物，中间速度提取最适合的β-连环蛋白降解。我nterphase提取物同样强大的促进β-catenin降解虽然是劳动密集型的准备。 <ol><li>加亮抑酶肽，胃蛋白酶抑制剂，抑酶肽混合物（LPA，蛋白酶抑制剂），以10微克/毫升和细胞松弛素D（从10 mg / ml的储备液在DMSO中稀释）在20微克/毫升（从10 mg / ml的储备液稀释在DMSO）为剩余的20毫升0.1倍的MMR。 </li><li>添加0，X MMR含有LPA及松弛素D的冲鸡蛋，轻轻摇动，孵育5分钟，在16°C。 </li><li>转蛋放入16℃下预冷的50ml离心管中，使蛋定居，并从顶部除去残留的缓冲区。为了防止暴露在空气中，提取少量的缓冲进入移液管之前撤回鸡蛋转移。继续额外蛋转移到离心管中，从管的顶部除去残留的缓冲液，直到卵填充离心管的顶部，这将最大化的产率提取。 </li><li>使用一个固定角转子包蛋，旋离心管中，在400×g离心60秒，在4℃。从离心管的顶部除去残留的缓冲区。 </li><li>对于粉碎的旋转，旋转管以15,000×g离心5分钟，在4℃下</li></ol></li><li>收集提取的细胞质层 注意：下面描述的方法不同于穿刺离心管的侧面，以便收集胞质层的经典方法。此协议已被改编为简化和用于与特定的离心管中，这是可重复使用的用途。 β-catenin降解动力学无明显差异发现了这两种方法。在这点上提取应保持冷时在整个过程中，所有步骤应在4℃下进行<ol><li>清除在使用P1000的枪头脂质层的孔。 </li><li>收集细胞质层（暗色素层和里之间向右脂质层）使用一个新的P1000的枪头成清洁预冷的离心管中（ 图1）。供高品质的提取物，鲁棒降低β-连环蛋白，最大程度地减少被撤回的细胞质层着色和脂质层的量。 </li><li>自旋提取细胞质层，以15,000×g离心10分钟，在4℃，并再次收集细胞质层。重复旋转和提取1X。注：该提取物应该是“稻草色。”如果有大量的污染与颜料和脂质层在这一点上，可以重复该自旋再一次，尽管过多的自旋会降低提取物的降解β-连环蛋白的能力。 </li><li>添加LPA及松弛素D的提取物的终浓度为10μg/ ml的每个。注意：使用所描述的方法产生约50毫克/毫升的相当一致的总蛋白浓度（52.41±5.40毫克/毫升，n = 3的独立的Preps）在爪蟾如克提取物。 </li><li> （可选）对于翻译实验，加帽mRNA（0.1毫克/毫升），核糖核酸酶（1.5单位/毫升），和能源再生混合（2.2.1），并在室温下孵育2小时反应（有关详细信息，请参阅萨利克等人 2和西伯等人 33）。立即β-catenin降解实验或管理单元冷冻在液氮中，供以后使用使用翻译摘录。注：新鲜制备的提取物具有较高的容量，以翻译外源添加的mRNA，但不幸的是，这个容量丢失一次提取液被冻结。封顶的mRNA可以使用市售的试剂盒而容易地制备。 </li><li>卡入式冷冻萃取物在液氮中。注：提取存储在小（200微升）等分供单人使用，因为他们很快失去其降解β-连环蛋白，如果再冷冻能力。对于长期储存，提取物可以储存在液氮中。对于短期储存，提取物可以储存在-80℃，虽然容量Øf提取物降解β-连环蛋白可以具有持久的贮存急剧下降，在-80℃（超过2个月的时间）。 </li></ol></li></ol> 2，准备提取β-catenin的降解测定<ol><li>从非洲爪蟾提取枯竭 注： 非洲爪提取物的主要优点是容易消耗的途径的组分和精确地加回一个定义的蛋白质的量，以确定它的剂量依赖性效应的能力。 <ol><li>使用新鲜配制的爪蟾卵提取物或快速解冻冷冻提取物，置于冰上。执行在寒冷的所有操作。 </li><li>添加提取物的1/10沉淀抗体或亲和珠（ 例如 ，将20ml沉淀珠〜200毫升提取液）的体积。为了尽量减少稀释的提取物，用凝胶加载提示长，锥形尖端除了提取物之前从珠尽可能撤出尽可能多的液体。 </li><li>旋转提取物珠混合物在4℃下1小时。 </li><li>旋提取物珠组合在12600 XG在离心在4℃下30秒。另外，如果磁性珠的使用，应用磁场来收集珠。 </li><li>转让贫提取物在冰上新的试管。要小心，不要将任何珠提取物。 </li><li>通过免疫印迹既耗尽提取物和珠子确认枯竭的效率。 </li><li>在2.2的介绍准备提取物β-catenin降解试验。 </li></ol></li><li> 非洲爪蟾优化提取物β-catenin的降解 注：在非洲爪蟾卵提取物β-连环蛋白的降解是一个能源依赖的过程，消耗较快的内源性ATP的商店。因此，能源再生系统必须保持强大的β-catenin降解。 <ol><li>准备一个20倍的能量再生（ER）混合组成的150 mM的磷酸肌酸，20毫摩尔ATP，600微克/毫升肌酸磷酸激酶，和20毫米MgCl 2。 ER应等分并储存于-80℃。通过使用小型冷冻分装，避免反复冷冻/解冻周期。 </li><li>摩擦双手之间的冷冻管迅速解冻爪蟾卵提取物。将试管置于冰上之前所有提取物的融化。 </li><li>加入10微升再生能源组合（20X ER）的成爪蟾卵提取物等分（200微升）。通过快速轻弹管和脉冲涡流调匀。脉冲自旋，并立即放置冰上。 </li><li> （可选）β-连环蛋白的营业额可以稍微加入泛素（1.25毫克/毫升决赛）增强在非洲爪蟾卵提取物。放线菌酮（0.1 mg / ml的终）也可以被添加，以减少内源性转录本的翻译。 </li><li>分装相应的卷对降解测定到在冰上预冷的微量离心管中。对于放射性标记的β-连环蛋白降解测定中，撤回2-5毫升提取每个时间点。&lt;/ LI&gt; </ol></li></ol>在非洲爪蟾卵提取物3。放射性标记β-catenin降解法注意：所有的操作步骤应在冰上进行，除非另有说明。 <ol><li>准备放射性标记β-连环蛋白<ol><li> 在使用市售的试剂盒体外合成的[35 S]甲硫氨酸的放射性标记的蛋白制备新鲜。注：生成35 S-标记的（半衰期87天）的蛋白质是使用市售的体外偶联转录翻译试剂盒很容易地和有效地生产。重要的是，在翻译的蛋白被充分地标记的，使得变化的蛋白质周转可以容易地可视化。 </li><li>确认成功的放射性标记，用0.5微升翻译的蛋白进行SDS-PAGE/autoradiography。量化放射性标记的β-连环蛋白带的使用ImageJ，ImageQuant，或另一种定量软件程序控制的强度是。注：放射性标记的β-连环蛋白的蛋白条带应该是在胶片上清晰可见几个小时（4-6小时）内。 </li><li>管理单元冷冻在液氮中的放射性标记的蛋白质储存直至使用。注意：长时间储存（&gt; 2个月）和多个冷冻/解冻（大于2）可以严重影响放射性标记的β-连环蛋白在非洲蟾蜍卵提取物鲁棒降解的能力。通常情况下，放射性标记的蛋白质的稳定性储存1个月，在-80℃，之后显著下降;因此，比较新鲜制备放射性标记的β-连环蛋白给出了在这些降解测定法最好的结果。 </li></ol></li><li>进行β-连环蛋白降解测定<ol><li>加体外翻译的β-连环蛋白的1-3微升（取决于放射性标记的频带信号的强度）（和其他蛋白质，小分子等，它们正在测试中）到20微升的非洲爪蟾的反应混合物在冰上。通过quickl调匀Ÿ轻弹管和涡旋的短脉冲;这是一个重要的步骤，因为爪蟾卵提取物很粘稠，并充分搅拌会影响结果的一致性。脉冲旋转，置于冰上。 </li><li>开始通过管转移到RT的β-连环蛋白降解反应。 </li><li>在指定的时间点，取出1-5毫升的样品，用SDS样品缓冲液（5倍体积）立即混合以终止反应。为了确保降解反应完全终止，轻弹管数次和涡大力。 </li><li>执行SDS-PAGE/autoradiography。运行1毫升当量的提取物（〜50毫克的蛋白质），每个时间点/通道。 β-连环蛋白在非洲蟾蜍卵提取物的劣化，应通过时间依赖性降低的放射性标记的β-连环蛋白带如图2的强度来证实。使用ImageJ，ImageQuant，或其它优选的成像软件，如果必要的量化结果。 </li><li> （OPTIONAL）浸泡SDS-聚丙烯酰胺凝胶在干燥前固定溶液（10％乙酸和30％甲醇的蒸馏水）以降低背景放射性和提高图像的质量。 </li></ol></li></ol> 4，β-连环蛋白 - 荧光素酶在非洲爪蟾卵提取物降解测定执行在冰上的所有步骤，除非另有说明。 <ol><li>准备β-连环蛋白 - 荧光素酶<ol><li>利用转录 - 翻​​译偶联系统与完整的氨基酸组合合成非放射性标记，荧光素酶标记的β-连环蛋白。 </li><li>确认生产的荧光素酶标记的β-连环蛋白通过测量荧光素酶活性从0.5-1微升反应的。通过从非翻译反应混合物测量发光评估背景发光。注：多个商业试剂盒可用于测量荧光素酶活性。长寿命的发光，然而，尤其有效的降解阿萨年。 </li></ol></li><li>进行β-连环蛋白 - 荧光素酶降解测定<ol><li>解冻并准备爪蟾卵提取物为2.2。 </li><li>加入体外翻译的β-连环蛋白-荧光素酶融合（4.1）到非洲爪蟾制备反应混合物（2.2）在冰和混合以及在3.2.1。注意：被添加到提取物中的β-连环蛋白的荧光素酶的活性通常在20 - 50,000相对发光单位提取物（RLU）/毫升（基于从4.1.2获得的测量结果）。启动信号应约100,000 RLU（2-5毫升体外翻译的β-连环蛋白-荧光素酶融合）。 </li><li>按住Shift键提取到RT 开始降解反应。 </li><li>去除反应的等分试样在指定的时间和管理单元冷冻在液氮中。注意：一式三份样品通常除去用于分析每个时间点。冷冻提取物可以贮存于-80℃下 UNTIL他们已经准备好进行分析。 </li><li>解冻样品冰，转移样本，以标准白色96孔板中在冰上，并处理为荧光​​素酶活性。 </li></ol></li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

爪蟾卵提取物是一种强大的生化系统的研究β-catenin的营业额。 β-连环蛋白在非洲蟾蜍卵提取物的浓度为〜25nM的2。在最佳条件下，卵提取物是能够在50-100纳米/小时的速度降低β-连环蛋白，并且是半最大，在200 nM的24。有β-catenin的降解利用非洲爪蟾卵提取物成功重建的几个关键步骤。这些包括：1）生成高品质的爪蟾卵提取物和由卵提取物的制备方式，2?...

A correction was made to <a href="http://www.jove.com/video/51425">Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract</a>. At the time of publication there were some instances where an incorrect volume notation was used. These instances were corrected from:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 ml pelleted beads to 200 ml extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 ml extract for each time point.
3.2.3. At the designated time point, remove 1-5 ml of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 ml equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/ml of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 ml of the in vitro-translated &#946;-catenin-luciferase fusion).
to:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 &#181;l pelleted beads to 200 &#181;l extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 &#181;l extract for each time point.
3.2.3. At the designated time point, remove 1-5 &#181;l of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 &#181;l equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/&#181;l of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 &#181;l of the in vitro-translated &#946;-catenin-luciferase fusion).

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

非洲爪蟾卵提取物已被广泛用于研究许多细胞生物学过程，包括细胞骨架动力学，核组装和进口，细胞凋亡，泛素代谢，细胞周期进程，信号转导和蛋白质周转1-17。在爪蟾卵提取系统是适合于细胞过程的一个军团的生化分析，因为卵提取物本质上代表未稀释的细胞质包含所有必要执行这些过程，使调查必要的细胞质成分。大量的卵提取物中可同时适用于需要大量的材料（ 例如 ，蛋白纯化或高通量筛选）18-20生化操作做好准备。另一个优点是，在非洲爪蟾卵提取物的特定蛋白质的浓度可以通过加入重组蛋白和/或endog的免疫耗竭的精确调整源性蛋白质相反的质粒DNA，其中感兴趣的蛋白质的表达是难以控制的转染。此外，由于缺乏可用的重组蛋白可通过加入转录编码目的蛋白质的被克服，取新鲜制备的非洲爪蟾卵提取物的高容量翻译外源添加的mRNA的优势。 蛋白质降解的调控是许多细胞途径的关键控制和处理21。 爪蟾卵提取物已被广泛用于研究蛋白质降解为系统允许多个方法来监测蛋白质周转不转录和翻译的混杂影响。 Wnt信号通路是一个高度保守的信号传导途径中起着发育和疾病的关键作用。 β-连环蛋白，Wnt信号通路的主要效应的营业额，受到严格的监管，并增加稳态升β-连环蛋白的伊维尔基尼是对Wnt靶基因的活化是至关重要的。 β-连环蛋白降解的重要性通过以下事实，在Wnt途径的突变抑制β-连环蛋白降解发现在〜90％的结直肠癌22都散发病例的高亮显示。通过Wnt信号通路的组成部分β-catenin降解，可以忠实地概括在非洲爪蟾卵提取物，研究其营业额的机制，以及确定其降解2，19，20，23-29的新型小分子调节剂。 方法，用于制备非洲爪蟾卵提取物用于研究细胞周期的前一朱庇特的出版物30-32进行了描述。当前协议描述了这些方法的变型，并且对降解的优化[35 S] -放射标记的β-连环蛋白和荧光素酶标记的β-连环蛋白在爪蟾卵提取物。放射性标记的降解测定允许蛋白水平的放射自显影通​​过直接观察。 [35 S]甲硫氨酸掺入到感兴趣的地方使用，然后可以直接添加到降解反应在体外翻译反应的蛋白质。此外，放射性标记的蛋白质周转测定法不需要对感兴趣的蛋白质或表位标签，这可以影响蛋白质稳定性的抗体。因为在蛋白水平的微小变化，这反映在变化放射性标记的蛋白条带的强度，很容易放射自显影时，[35 S]-放射性标记的降解测定代表蛋白质周转2的可视化一个非常有用的方法。 β-连环蛋白，以萤火虫荧光素酶的融合（以下简称为“荧光素酶”）允许对蛋白水平的精确和轻便的定量测量，以来确定β-连环蛋白周转19,20的动力学性质。萤光素酶测定法的一个主要优点是它提供了一个强有力的定量系统，很容易按比例放大。下面的协议提供了简单的方法，用于测定β-连环蛋白降解和健壮，高效且有效的方法的新的β-连环蛋白调节剂的高通量筛选。

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			<td height="42" style="height:42px;width:216px;">Pregnant Mare Serum Gonadotropin (PMSG)</td>
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			<td>Reconstituted with distilled water before use.&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Human Chorionic Gonadotropin</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Chloride</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:119px;">BP366-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride</td>
			<td style="width:132px;">Research Products International</td>
			<td style="width:119px;">S23020-5000.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:119px;">BP214-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium Chloride</td>
			<td style="width:132px;">Acros Organics</td>
			<td style="width:119px;">AC42352-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:119px;">BP310-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cysteine</td>
			<td style="width:132px;">Acros Organics</td>
			<td style="width:119px;">AC17360-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leupeptin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">L2884-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aprotinin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">A1153-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pepstatin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">P4265-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytochalasin B</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">C8273-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 mL syringe: Luer Lock tip</td>
			<td style="width:132px;">Becton Dickinson</td>
			<td style="width:119px;">309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">27G1 needle</td>
			<td style="width:132px;">Becton Dickinson</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96 well solid white polystyrene microplate, round bottomed</td>
			<td style="width:132px;">Corning</td>
			<td style="width:119px;">3605</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Steady-Glo Luciferase assay system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">E2520</td>
			<td>Store long-term at -80, can store for up to 1 month at -20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TNT Sp6 coupled reticulocyte lysate system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">L4600</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TNT Sp6 High-yield Wheat Germ protein expression system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">L3260</td>
			<td>Generally higher yield than reticulocyte lysate&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EasyTag Express Protein Labeling Mix [S35]</td>
			<td style="width:132px;">Perkin Elmer</td>
			<td style="width:119px;">NEG772007MC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine phosphate</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">27920-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ATP</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">A2383-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine phosphokinase</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">C3755-35KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">D8418-50ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dual-Glo luciferase assay system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">E2920</td>
			<td>Same storage conditions as Steady-Glo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL Centrifuge tubes</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">0556214D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sorvall SS-34 fixed angle rotor</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:119px;">28020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">115 V 50/60 Hz Minicentrifuge</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">05-090-128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mMessage mMachine Sp6 kit</td>
			<td style="width:132px;">Ambion</td>
			<td style="width:119px;">AM1340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Firefly Luciferase antibody</td>
			<td style="width:132px;">Abcam</td>
			<td style="width:119px;">ab16466</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-GSK3 antibody</td>
			<td style="width:132px;">BD Transduction Laboratories</td>
			<td style="width:119px;">610201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FLUOStar Optima</td>
			<td style="width:132px;">BMG Labtech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sorvall RC-6 Plus Centrifuge</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16&#176;C Incubator</td>
			<td style="width:132px;">Percival Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of &#946;-catenin degradation in xenopus egg extract

非洲爪蟾卵提取物是一种良好的特点，强大的系统为研究不同细胞过程的生物化学。 爪蟾卵提取物已被用于研究蛋白质周转的许多细胞环境，包括细胞周期和信号转导通路1-3。在此，描述了一种方法用于隔离爪蟾卵提取物进行了优化，以促进关键Wnt信号通路组分，β-连环蛋白的降解。两种不同的方法进行了说明，以评估在非洲爪蟾卵提取物β-连环蛋白降解。一种方法是在视觉信息（[35 S] -放射性标记的蛋白质），而另一种是更容易按比例用于高通量测定法（萤火虫荧光素标记的融合蛋白）。所描述的技术可用于，但不限于，评估β-连环蛋白周转和识别分子组分贡献的营业额。此外，快速的学习性净化大量的同质爪蟾卵提取物结合荧光素标记的蛋白质的定量和轻便读出允许这个系统可以很容易地适用于高通量筛选β-catenin降解的调节剂。

在非洲爪蟾卵提取物β-连环蛋白降解的示意图示于图2A中 。35 S-标记的β-连环蛋白孵育在非洲爪蟾卵提取物，等分试样（1毫升提取当量）除去在适当的时间，并且样品进行SDS-PAGE后的放射自显影。通过Wnt通路的组分β-连环蛋白降解是通过遍在蛋白-蛋白酶体系统2介导的和[35 S] -放射标记的β-连环蛋白在非洲爪提取物的降解通过加入蛋白酶体?...

Watch this Scientific Journal Video about Reconstitution Of Î²-catenin Degradation In Xenopus Egg Extract at JoVE.com

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

的方法被描述为使用放射性标记和荧光素酶融合蛋白在非洲蟾蜍卵提取物和其适应高通量筛选用于蛋白质降解的小分子调节剂进行分析的蛋白质降解。

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 ...

reconstitution-of-catenin-degradation-in-xenopus-egg-extract

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

Unit 1

Translation: RNA to Protein

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12.0K 次观看.  Vanderbilt University Medical Center. 的方法被描述为使用放射性标记和荧光素酶融合蛋白在非洲蟾蜍卵提取物和其适应高通量筛选用于蛋白质降解的小分子调节剂进行分析的蛋白质降解。 

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

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

科研

生物学

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

研究DNA损伤检验点<em&gt;爪蟾</em&gt;卵提取物

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

口面表型的定量<em&gt;爪蟾</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).