这项工作是由英国心脏基金会的奖学金，以SZ（FS / 08/063和FS / 15 /三万一千四百九十四分之三十○）的支持。

1.酵母双杂交<ol><li> 酵母转化 <ol><li>准备下列媒体和缓冲区: <ol><li>通过将20克/升胨，10克/升酵母提取物，2％w / v的葡萄糖（高压灭菌后加入）和20g / L琼脂制备酵母完整酵母提取物 - 蛋​​白胨 - 葡萄糖（YPD）培养基（对于板只） ;通过高压灭菌消毒并在实验当天用新鲜。 </li><li>准备酵母最小的合成定义（SD）中混合6.7克/酵母氮基L的，​​1.6克/升降补充缺乏亮氨酸和色氨酸，2％W（缺乏亮氨酸和色氨酸，以保持两个诱饵和目标质粒选择压力） / v的葡萄糖（高压灭菌后加入）和20g / L琼脂（对于板只）;通过高压灭菌消毒并在实验当天用新鲜。 </li><li>制备50％w / v的PEG（聚乙二醇）3350;通过0.2微米的过滤器，并存储在RT消毒。 </li><li>准备100毫米的Tris / 10毫米EDTA（TE 10倍），调整pH值至7.5，灭菌通过0.2微米的过滤器和储存在室温。 </li><li>制备的1M乙酸锂（10×醋酸锂）;将pH调节至7.5，用CH 3 COOH，通过0.2微米的过滤器，并存储在RT消毒。 </li></ol></li><li>划线冷冻甘油股票少量到YPD板重振Y2H株（如 Y190）。在30℃下孵育直至酵母菌落达到〜2毫米的直径（通常为3 - 5天，这取决于酵母菌株）。 </li><li>接种0.5毫升YPD的（在1.5ml管）与一个单一的，大的（2 - 3毫米直径）菌落。涡大力为〜2分钟驱散任何团块。 </li><li>转移细胞悬液至含有50ml YPD培养基的500ml烧瓶中。在30℃下为16孵育 - 18小时以250rpm对于酵母摇动达到静止期。 </li><li>转移4 -过夜培养5毫升入200ml YPD中（在1升锥形瓶中），以在600nm（OD 600产生的光密度，测定使用0.2分光光度计） - 0.3（200毫升将足以为20变换）。 </li><li>在30℃下孵育以250rpm振荡培养直至细胞在对数中期， 即 ，外径600 = 0.5 - 0.6（通常在2 - 3小时）。 </li><li>收获酵母经离心（在50ml试管）在1500 xg离心在RT 5分钟。弃去上清液，在5毫升无菌H 2 O和游泳池的悬浮颗粒每一起。 </li><li>重新离心机以1,500×g离心在RT 5分钟并弃上清。悬浮颗粒酵母1毫升新鲜配制，无菌1X醋酸锂/ TE的。 注:准备1小时内使用主管酵母细胞。 </li><li>通过添加对单个变换200纳克质粒DNA，或0.5制备质粒样品（在1.5ml试管） - 1每种质粒DNA用于共转化的微克，以及100微克鲱鱼睾丸载体DNA（煮沸20分钟并冷却上使用冰前夕）。 注:加入的阳性对照， 例如 ，酵母。共转化PVA3（编码GAL4的DNA-BD融合与p53蛋白）和PTD1（编码与SV40大T抗原GAL4 AD融合）。 </li><li>添加新鲜制备的，主管酵母悬浮液（步骤1.1.8）的100微升和600微升的1倍醋酸锂/聚乙二醇溶液（8毫升库存PEG 3350 1 ml储备的TE中，加入1ml的库存醋酸锂的），和涡旋〜30秒。孵育在30℃下进行30分钟并以200rpm振荡。 </li><li>添加80微升二甲亚砜（10％体积/体积终浓度）并轻轻地倒转混合。在混合的同时在42℃水浴中15分钟热冲击，每2 - 3分钟。 </li><li>冰浴细胞悬浮液2分钟并离心在14,000rpm xg离心15秒在RT恢复酵母。 </li><li>在100微升TE 1X的悬浮细胞沉淀和板适当的最小SD培养基平板上选择生长（培养基缺乏亮氨酸和色氨酸，以保持两个诱饵和目标质粒选择压力）。 </li><li>孵育板向上面朝下，在30°C，直到菌落〜2毫米直径（一般为4 - 5天）。板可在4℃下2 - 3周;更长的存储使甘油股票并储存在-80℃。 注意:验证诱饵和目标蛋白质在酵母中表达的免疫印迹17，并且它们不具有自主报告基因活化酵母（如在第1.3节描述的β-Gal的测定）时分别表示。 </li></ol></li><li> 菌落电梯滤纸β-Gal的含量 <ol><li>准备以下缓冲区: <ol><li>制备含有100mM 的 Na 2 HPO 4，Z缓冲器40毫的NaH 2 PO 4，10mM的氯化钾，1mM的MgSO 4上;将pH调节至7.4。通过高压灭菌和储存在室温下消毒。 </li><li>通过将制备的X-gal缓冲5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷在-20℃20毫克/毫升的N，N-二甲基甲酰胺和存储在黑暗中。准备Z缓存/ X-Gal的解决方案。使缓冲区通过混合的X-gal在0.33毫克/毫升和β巯基乙醇0.27％体积/体积的Z缓冲器使用前。每个采样使用2.5毫升。 </li></ol></li><li>加入2.5倍毫升一个干净的100毫米的钢板和地点纤维素滤纸内新鲜配制的Z缓存/ X-Gal的解决方案。 </li><li>将新的滤纸上的板的表面上与待测定的酵母菌落。轻轻揉搓滤纸上钳板，并留下〜5分钟菌落附着。 注:并行处理阳性对照， 即 ，酵母共转化PVA3和PTD1。 </li><li>提起滤纸和淹没它（用钳子）到液氮30秒池（液氮应小心处理;总是穿厚厚的手套和护目镜）。让我们在RT冷冻滤纸解冻〜2分钟。 </li><li>放置在100毫米的钢板内的预先浸泡的滤纸顶部滤纸（菌落面朝上），并且在30℃下孵育。 </li><li>定期检查（每〜30分钟）为蓝色菌落的外观。酵母Y190共转化与阳性对照质粒（PVA3 + PTD1）将变成在60分钟（未发表的观测值）为蓝色。 注:弱诱饵:靶相互作用可能需要几个小时，产生了积极的蓝色信号（避免长时间的潜伏期（&gt; 8小时），可能会给假阳性结果）。为获得最佳效果，可以使用新鲜的共转化的菌落， 即 ，在30℃下生长4 - 5天，〜2毫米的直径。 </li></ol></li><li> 液体培养β-Gal的含量 <ol><li>准备以下缓冲区: <ol><li>制备含有100mM 的 Na 2 HPO 4，Z缓冲器40毫的NaH 2 PO 4，10mM的氯化钾，1mM的MgSO 4上;将pH调节至7.4，在室温下通过高压灭菌和储存消毒。 的1M 的 Na 2 CO 3;储存在室温。 </li><li>准备Z缓存/β巯基乙醇。使通过添加β巯基乙醇0.27％对使用缓冲之前/V IN Z缓存;每个采样使用700微升。准备Z缓存/ ONPG解决方案。使前缓冲器通过混合ONPG（邻 - 硝基苯基β-D-吡喃半乳糖苷），在4毫克/毫升和β巯基乙醇0.27％在Z缓冲器体积/体积使用;每个采样使用160微升。 </li></ol></li><li>使用单菌落接种5毫升最小的SD培养基（缺乏亮氨酸和色氨酸，以保持在两个诱饵和目标质粒的选择压力），并在30℃下为16孵育 - 18小时以250rpm摇动。 注:测定五个单独的菌落共转化使用相同的诱饵和目标质粒。 </li><li>传输足够的过夜培养物在10ml新鲜的SD培养基中，以产生的OD 600 = 0.2 - 0.3。在30℃下孵育以250rpm振荡培养直至细胞在对数中期（OD 600 = 0.5 - 0.6）。 </li><li>转移0.5毫升酵母培养到1.5毫升管和离心机在14,000rpm xg离心2分钟，在室温。收获时，CE记录确切的OD 600LLS。悬浮颗粒在Z缓存100μl的;这将导致在5倍浓度的因子。 </li><li>在液氮地方管约1分钟（液氮应小心处理;总是穿厚厚的手套和护目镜），然后在37℃水浴3分钟解冻。重复此冻融循环两次，以确保细胞被破开。 </li><li>设置用100μlZ缓冲器的管坯。 </li><li>添加Z缓冲器/β巯基乙醇700微升和160微升Z缓冲器/ ONPG到样品和空白管;开始在30℃培养箱定时器和地点。 </li><li>定期检查黄色发展。加入400微升，1M的Na 2 CO 3的停止显色，并记录经过的时间（分钟）。酵母Y190共转化与阳性对照质粒（PVA3 + PTD1）将在60分钟内变为黄色.. 注:弱诱饵:靶相互作用可能需要几个小时，产生了积极的黄色信号，为获得最佳重sults，使用新鲜的共转化的菌落， 即 ，在30℃下进行4生长- 5天，〜2毫米的直径。 </li><li>离心在14,000rpm xg离心5分钟，在室温以沉淀细胞碎片，将上清液转移到一个干净的试管中。 </li><li>使用分光光度计，测量在相对 ​​于空白（值应为0.02之间- 1.0）样品的420纳米（A 420）的吸收。 </li><li>计算β半乳糖苷酶单位，定义为水解ONPG的1微摩尔至邻硝基苯酚和每细胞分钟D-半乳糖的量1单位，按下列公式计算: <img alt="式（1）" src="/files/ftp_upload/54271/54271eq1.jpg" /> =β半乳糖苷酶单位 其中:T:孵化的时间（以分钟为单位）;比照:从步骤1.4.8， 即集中系数，CF = 5; OD 600:收集细胞时。 </li></ol></li></ol> 2.在哺乳动物细胞系蛋白质表达<ol><li><st荣&gt;哺乳动物细胞转染<ol><li>准备下列媒体和缓冲区: <ol><li>通过用4.5克/升葡萄糖，10％体积/体积的胎牛血清和2mM L-谷氨酰胺的DMEM混合制备生长培养基;通过0.2微米的过滤器和储存在4℃下灭菌。 </li><li>通过混合280毫摩尔NaCl，10毫氯化钾，制备2×HEPES-缓冲盐水（2×HBS）1.5毫的 Na 2 HPO 4，12mM的葡萄糖，50毫摩尔的Hepes;将pH调节至7.05，通过0.2微米的过滤器，并储存在-20℃下灭菌。通过0.2微米的过滤器和储存在-20°C准备2.5M的氯化钙2，灭菌。 </li></ol></li><li>转染前一天，种子1 - 2×10 6个 HEK293细胞在100毫米培养皿中，以便60-70％汇合翌日。培养16 - 18 5％的CO 2的潮湿培养箱中于37℃小时。 </li><li>在转染的当天，除去旧的介质和进料的细胞用10毫升新鲜生长培养基中。 注意:抗生素从培养基转染期间省略，因为它们可能会增加细胞死亡。 </li><li>稀释24微克质粒DNA（用于共转染时，两种质粒的等摩尔比），在600微升总体积60微升的2.5M 的 CaCl 2 1.5ml试管内（用无菌去离子水2 O制成）;旋涡混匀。 注:对于转染效率最高，质粒DNA应该是最高的纯度， 即 ，有一个绝对260 / ABS 280比= 1.8〜。 </li><li>添加质粒DNA /钙溶液滴到含有600微升2×HBS的同时不断大力通过涡旋混合50毫升管中。孵育20分钟，在RT至允许形成磷酸钙/质粒DNA复合物。 </li><li>在20分钟温育后，涡旋简要后和明智的添加溶液滴到细胞上以覆盖100毫米的培养皿的整个表面面积。 </li><li>孵育所述细胞在37℃在5％CO <sUB&gt; 2。 〜6小时后更换生长培养基，并放回孵化器。 </li><li>收获离心所述细胞24小时转染后在1000 xg离心在RT 3分钟，并弃去上清液。细胞沉淀可直到需要被储存在-80℃。 注:通常表达峰24 - 72小时后转染。 </li></ol></li><li> 同质化的细胞 <ol><li>准备以下缓冲区: <ol><li>通过混合150毫米氯化钠，20毫米的Tris准备联合IP同质化的缓冲区;将pH调节至7.4，并在4​​℃下储存。 </li><li>制备通过混合5毫摩尔的Hepes，0.3M蔗糖交联匀浆缓冲液;将pH调节至7.4，并在4​​℃下储存（在使用前过滤器，以除去任何颗粒）。在使用前蛋白酶抑制剂补充。 </li></ol></li><li>加入250微升的玻璃珠（425 - 600微米）1.5 ml的管中，并用500μl匀浆缓冲液中洗涤。一个简短的离心颗粒玻璃珠脉冲（1000 XG 5秒），去除上清。重复此清洗步骤一次。 </li><li>重悬细胞沉淀（从步骤2.1.8）于500μl冰冷均质化缓冲液中，将细胞悬液转移到含有珠的玻璃管。 </li><li>通过细针连接于1ml注射器20代（23克，0.6×30mm的）匀浆在冰上的细胞。与管帽封闭，刺穿通过它与针，并通过玻璃珠剧烈分散的细胞悬浮液。 </li><li>离心机在4℃下1500×g离心10分钟以除去细胞核和不间断的细胞，弃去沉淀。保存较后核部分上清，直接进行合作，IP或交联为宜。 </li></ol></li></ol> 3. 在体外生化方法<ol><li> 共免疫沉淀 <ol><li>准备以下缓冲区: <ol><li>准备联合IP同质化缓冲区s中描述2.2.1.1挠度。通过混合的20mM Tris，150 mM氯化钠，0.5％制备的IP缓冲液（重量/体积）CHAPS，2mM的二硫苏糖醇（可选;可以包括二硫苏糖醇，以减少可能形成因空气氧化蛋白质聚集）;将pH调节至7.4，并在4​​℃下储存。 </li><li>准备2％W /在合作同质化IP缓冲v CHAPS;存放于4℃。 </li><li>通过混合60毫摩尔Tris，2％w / v的SDS，10％体积/体积的甘油，5mM的EDTA，0.01％w / v的溴酚蓝，2％体积/体积β巯基乙醇（可选）制备蛋白 - 样缓冲液;调节在室温pH为6.8和存储。 </li></ol></li><li>从汇合百毫米培养皿均质化细胞（〜如HEK293，与使用血球的计数8×10 6个细胞），如第2.2节中描述。 </li><li>溶解在4℃下以恒定混合有0.5％CHAPS为≥2小时后核亚细胞级分（使用2％股票）和温育。离心机以20,000×g离心10分钟，在4℃以沉淀不溶马泰人弃沉淀。保存上清称为细胞裂解液。 注:洗涤剂和除去不溶性物质的夹杂物是绝对必要的，以减少非特异性结合。中间体洗涤剂， 例如 ，CHAPS或Triton X-100以0.2的浓度- 1％，是最常用的。 </li><li>准备两个独立1.5毫升管，并添加约20微升（取决于IgG的结合能力）6毫克/毫升蛋白A或蛋白G琼脂糖（或琼脂糖）珠。用200微升IP缓冲液清洗。 注:选择取决于用于提高免疫沉淀抗体的动物物种的相应的Ig结合树脂。蛋白G相比，蛋白A免疫球蛋白结合亚型的范围更广。 </li><li>离心1500 xg离心回收珠子在4℃下2分钟。重复洗具IP缓冲一次，然后在200微升IP缓冲区的悬浮颗粒。 </li><li>添加免疫沉淀抗体或非免疫的IgG 1微克（5毫微克/微升）（以服务于两个单独的蛋白A / G含有管作为阴性对照）。孵育在4℃下以恒定混合≥2小时。 注:始终处理与使用非免疫IgG的相同的动物物种的免疫沉淀抗体升高阴性对照。 </li><li>在4℃下以1500 xg离心离心回收珠子2分钟并小心地丢弃通过吸液上清。 </li><li>转移200μl的细胞裂解物（来自步骤3.1.3）的成每两个管与抗体和蛋白A / G珠粒。孵育≥2小时，在4℃下持续混合，以允许抗原 - 抗体结合。 </li><li>恢复珠，并用200微升IP缓冲洗净;在混合孵育10分钟，在4℃。回收珠子和洗两次以上（避免多次洗涤这将减少特定和非特异性两者结合）。小心丢弃吹打上清。 </li><li>添加30μl的蛋白加载缓冲液以洗脱immunoprecipitated蛋白质。离心机在4℃下1500×g离心2分钟，弃去珠子和保存含有洗脱共同的IP样品上清液。 </li><li>来验证成功的X蛋白沉淀，加载到用特异于蛋白质X的抗体进行免疫印迹17来分析在SDS-PAGE凝胶的共IP样品少量（1/10，3微升）中包括的等分试样细胞裂解液验证您的样品中X蛋白的表达。 </li><li>为了测试共沉淀的蛋白Y的存在，加载在一个单独的SDS-PAGE凝胶的共IP样品的其余部分（9/10 个 ，27微升）以通过与特定的蛋白质Y.抗体进行免疫印迹17进行分析包括细胞裂解物，以验证您的样品中蛋白质表达Ÿ一等分。 </li></ol></li><li> 化学交联 <ol><li>准备以下缓冲区: <ol><li>如在节2.2.1.1中所述的交联匀浆缓冲液。 </li><li>准备5倍p通过混合300毫摩尔Tris，10％w / v的SDS，50％体积/体积的甘油，25毫摩尔EDTA，0.05％w / v的溴酚蓝，10％体积/体积β巯基乙醇（可选）rotein加载缓冲器;调节在室温pH为6.8和存储;在温暖使用前50℃。 </li></ol></li><li>从汇合百毫米培养皿均质化细胞（〜如HEK293，与使用血球的计数8×10 6个细胞），如第2.2节中描述。 注意:蔗糖（在0.3M）用于创建一个等渗缓冲。盐， 例如 ，120 - 150毫摩尔NaCl或KCl可代替使用，这取决于所感兴趣的寡聚蛋白复合物。 </li><li>离心后核子亚细胞级分以20,000 xg离心10分钟，在4℃以沉淀蛋白质的聚集体，并保​​存上清液。以每20微升（通常是蛋白质的〜20微克）八等分成独立0.5毫升管。 </li><li>添加0.0025％体积/体积戊二醛所有样品并启动定时器。让每八个样品与glutarald反应在RT ehyde为:0，2，5，10，15，20，30，60分钟。 注:戊二醛具有与游离胺反应两个醛基。避免使用pH缓冲剂或其它物质与伯氨基，因为它们将猝灭戊二醛反应。 </li><li>停止戊二醛反应在含有2％体积/体积的肼在指定的时间，并添加5×蛋白加样缓冲液的5微升，使蛋白质变性。 </li><li>继续进行SDS-PAGE和免疫印迹17。 </li></ol></li></ol>

<ol><li>Seidel, M., Lai, F. A., Zissimopoulos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+functional+interactions+within+ryanodine+receptor%5BTitle%5D)+AND+%22Biochem+Soc+Trans%22%5BJournal%5D)">Structural and functional interactions within ryanodine receptor.</a> Biochem Soc Trans. 43 (3), 377-383 (2015).</li>
<li>Zissimopoulos, S., Marsh, J., Stannard, L., Seidel, M., Lai, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amino-terminus+oligomerisation+is+conserved+in+intracellular+calcium+release+channels%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Amino-terminus oligomerisation is conserved in intracellular calcium release channels.</a> Biochem J. 459 (2), 265-273 (2014).</li>
<li>Zissimopoulos, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-terminus+oligomerization+regulates+the+function+of+cardiac+ryanodine+receptors%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">N-terminus oligomerization regulates the function of cardiac ryanodine receptors.</a> J Cell Sci. 126 (Pt 21), 5042-5051 (2013).</li>
<li>Seidel, M., Thomas, N. L., Williams, A. J., Lai, F. A., Zissimopoulos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dantrolene+rescues+aberrant+N-terminus+inter-subunit+interactions+in+mutant+pro-arrhythmic+cardiac+ryanodine+receptors%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Dantrolene rescues aberrant N-terminus inter-subunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors.</a> Cardiovasc Res. 105 (1), 118-128 (2015).</li>
<li>Stewart, R., Zissimopoulos, S., Lai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligomerization+of+the+cardiac+ryanodine+receptor+C-terminal+tail%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Oligomerization of the cardiac ryanodine receptor C-terminal tail.</a> Biochem J. 376, 795-799 (2003).</li>
<li>Deane, C. M., Salwinski, L., Xenarios, I., Eisenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+interactions%3A+two+methods+for+assessment+of+the+reliability+of+high+throughput+observations%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Protein interactions: two methods for assessment of the reliability of high throughput observations.</a> Mol Cell Proteomics. 1 (5), 349-356 (2002).</li>
<li>Fashena, S. J., Serebriiskii, I., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+continued+evolution+of+two-hybrid+screening+approaches+in+yeast%3A+how+to+outwit+different+preys+with+different+baits%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits.</a> Gene. 250 (1-2), 1-14 (2000).</li>
<li>Stynen, B., Tournu, H., Tavernier, J., Van Dijck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity+in+genetic+in+vivo+methods+for+protein-protein+interaction+studies%3A+from+the+yeast+two-hybrid+system+to+the+mammalian+split-luciferase+system%5BTitle%5D)+AND+%22Microbiol+Mol+Biol+Rev%22%5BJournal%5D)">Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system.</a> Microbiol Mol Biol Rev. 76 (2), 331-382 (2012).</li>
<li>Yang, M., Wu, Z., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-peptide+interactions+analyzed+with+the+yeast+two-hybrid+system%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Protein-peptide interactions analyzed with the yeast two-hybrid system.</a> Nucleic Acids Res. 23 (7), 1152-1156 (1995).</li>
<li>Elion, E. A. Chapter 8, Detection of protein-protein interactions by coprecipitation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aElion%2C+EA+%22Curr+Protoc+Immunol%22">Curr Protoc Immunol</a>. , (2007).</li>
<li>Kingston, R. E., Chen, C. A., Rose, J. K. Chapter 9, Calcium phosphate transfection. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKingston%2C+RE+%22Curr+Protoc+Mol+Biol%22">Curr Protoc Mol Biol</a>. , (2003).</li>
<li>Lam, A. K., Galione, A., Lai, F. A., Zissimopoulos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hax-1+identified+as+a+two-pore+channel+%28TPC%29-binding+protein%5BTitle%5D)+AND+%22FEBS+letters%22%5BJournal%5D)">Hax-1 identified as a two-pore channel (TPC)-binding protein.</a> FEBS letters. , (2013).</li>
<li>Zissimopoulos, S., Lai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+FKBP12.6+with+the+cardiac+ryanodine+receptor+C-terminal+domain%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Interaction of FKBP12.6 with the cardiac ryanodine receptor C-terminal domain.</a> J Biol Chem. 280, 5475-5485 (2005).</li>
<li>Zissimopoulos, S., Thomas, N. L., Jamaluddin, W. W., Lai, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FKBP12.6+binding+of+ryanodine+receptors+carrying+mutations+associated+with+arrhythmogenic+cardiac+disease%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">FKBP12.6 binding of ryanodine receptors carrying mutations associated with arrhythmogenic cardiac disease.</a> Biochem J. 419 (2), 273-278 (2009).</li>
<li>Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfection+of+adherent+and+suspended+cells+by+calcium+phosphate%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Transfection of adherent and suspended cells by calcium phosphate.</a> Methods. 33 (2), 136-143 (2004).</li>
<li>Kaboord, B., Perr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+proteins+and+protein+complexes+by+immunoprecipitation%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation of proteins and protein complexes by immunoprecipitation.</a> Methods Mol Biol. 424, 349-364 (2008).</li>
<li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSambrook%2C+J+%22Molecular+cloning%3A+a+laboratory+manual%22">Molecular cloning: a laboratory manual</a>. , 2nd, Cold Spring Harbor Laboratory Press. (1989).</li>
<li>Yang, W., Steen, H., Freeman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+approaches+to+the+analysis+of+multiprotein+signaling+complexes%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Proteomic approaches to the analysis of multiprotein signaling complexes.</a> Proteomics. 8 (4), 832-851 (2008).</li>
<li>Migneault, I., Dartiguenave, C., Bertrand, M. J., Waldron, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutaraldehyde%3A+behavior+in+aqueous+solution%2C+reaction+with+proteins%2C+and+application+to+enzyme+crosslinking%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.</a> Biotechniques. 37 (5), 790-796 (2004).</li>
<li>Wine, Y., Cohen-Hadar, N., Freeman, A., Frolow, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elucidation+of+the+mechanism+and+end+products+of+glutaraldehyde+crosslinking+reaction+by+X-ray+structure+analysis%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis.</a> Biotechnol Bioeng. 98 (3), 711-718 (2007).</li>
<li>Zissimopoulos, S., Lai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+domain+of+the+human+cardiac+muscle+ryanodine+receptor+does+not+mediate+interaction+with+FKBP12.6%5BTitle%5D)+AND+%22Cell+Biochem+Biophys%22%5BJournal%5D)">Central domain of the human cardiac muscle ryanodine receptor does not mediate interaction with FKBP12.6.</a> Cell Biochem Biophys. 43, 203-220 (2005).</li>
<li>Zissimopoulos, S., West, D., Williams, A., Lai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ryanodine+receptor+interaction+with+the+SNARE-associated+protein+snapin%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Ryanodine receptor interaction with the SNARE-associated protein snapin.</a> J Cell Sci. 119, 2386-2397 (2006).</li>
<li>Zissimopoulos, S., Docrat, N., Lai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Redox+sensitivity+of+the+ryanodine+receptor+interaction+with+FK506-binding+protein%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Redox sensitivity of the ryanodine receptor interaction with FK506-binding protein.</a> J Biol Chem. 282, 6976-6983 (2007).</li>
<li>Zissimopoulos, S., Seifan, S., Maxwell, C., Williams, A. J., Lai, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disparities+in+the+association+of+the+ryanodine+receptor+and+the+FK506-binding+proteins+in+mammalian+heart%5BTitle%5D)+AND+%22J+Cell+Sci.+125%22%5BJournal%5D)">Disparities in the association of the ryanodine receptor and the FK506-binding proteins in mammalian heart.</a> J Cell Sci. 125. 125 (Pt 7), 1759-1769 (2012).</li>
<li>Ali, M. H., Imperiali, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+oligomerization%3A+how+and+why%5BTitle%5D)+AND+%22Bioorg+Med+Chem%22%5BJournal%5D)">Protein oligomerization: how and why.</a> Bioorg Med Chem. 13 (17), 5013-5020 (2005).</li>
<li>Hashimoto, K., Panchenko, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+protein+oligomerization%2C+the+critical+role+of+insertions+and+deletions+in+maintaining+different+oligomeric+states%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Mechanisms of protein oligomerization, the critical role of insertions and deletions in maintaining different oligomeric states.</a> Proc Natl Acad Sci U S A. 107 (47), 20352-20357 (2010).</li>
<li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Soluble+protein+oligomers+in+neurodegeneration%3A+lessons+from+the+Alzheimer%27s+amyloid+beta-peptide%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.</a> Nat Rev Mol Cell Biol. 8 (2), 101-112 (2007).</li>
<li>Chi, E. Y., Krishnan, S., Randolph, T. W., Carpenter, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physical+stability+of+proteins+in+aqueous+solution%3A+mechanism+and+driving+forces+in+nonnative+protein+aggregation%5BTitle%5D)+AND+%22Pharm+Res%22%5BJournal%5D)">Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation.</a> Pharm Res. 20 (9), 1325-1336 (2003).</li>
<li>Gell, D. A., Grant, R. P., Mackay, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+detection+and+quantitation+of+protein+oligomerization%5BTitle%5D)+AND+%22Adv+Exp+Med+Biol%22%5BJournal%5D)">The detection and quantitation of protein oligomerization.</a> Adv Exp Med Biol. 747, 19-41 (2012).</li>
<li>Kaczor, A. A., Selent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligomerization+of+G+protein-coupled+receptors%3A+biochemical+and+biophysical+methods%5BTitle%5D)+AND+%22Curr+Med+Chem%22%5BJournal%5D)">Oligomerization of G protein-coupled receptors: biochemical and biophysical methods.</a> Curr Med Chem. 18 (30), 4606-4634 (2011).</li>
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蛋白质同源低聚物的形成是一个基本的生物过程，调节转录因子，酶，离子通道和受体25,26的活性。重要的是，蛋白寡聚化也有病理的影响，包括神经功能障碍和心律失常的心脏疾病4,27。在这篇文章中所概述的方法用于识别域的域的相互作用介导蛋白自缔合和低聚。下面，我们指出在每个协议中的关键步骤，我们将讨论重要事项，限制和排除故障。 在Y2H系统可以采用先筛选，因为它的相对高通量筛选的潜在相互作用蛋白伙伴，易用性和高度可重复的结果。 Y2H过程中微生物实验室标准（板或振动筛），孵化器和室内封闭设施进行。采用FR的eshly制备的感受态细胞（步骤1.1.8）可以高效率酵母转化至关重要的，而对于在β-Gal的测定法最好的结果，新生长的酵母菌落（最多5天龄），应使用（步骤1.2.3）。基于不同于GAL4和/或附加的/替代报告基因等的转录因子的系统是可用的，并且因此诱饵和猎物质粒应当与适当的Y2H应变匹配。 一些菌株应该在3-氨基-1,2,4-三唑时，HIS3蛋白的竞争性抑制剂的存在下培养，以猝灭HIS3报道基因7,8-任何组成型表达。诱饵和靶融合蛋白质的表达应被免疫印迹17进行验证。万一诱饵/猎物融合蛋白是有毒的酵母，低级耐受的蛋白质水平可通过在诱饵/猎物表达是由不同的启动子驱动的不同载体克隆来实现。此外，重要的是要确保吨帽子诱饵/猎物融合蛋白不显示自主报告基因的活性。自主报告基因活化可以通过修改该构建以除去负责区域获救，或者通过交换与GAL4的DNA-BD和AD融合两个测试蛋白质。此外，跨膜结构域更好从诱饵/猎物构建省略，因为它们可引起细胞内的膜隔室错误折叠或融合蛋白的错误定位。的确，Y2H系统的主要缺点是，在诱饵和目标蛋白质在酵母核局部化从它们的生理亚细胞位置远的和潜在缺乏特定的翻译后修饰，从而产生假阳性或假阴性的相互作用6-9 。 哺乳动物的异源表达系统更适合于哺乳动物整合膜蛋白在构象，翻译后修饰和亚细胞定位方面的研究。其中最广泛使用的细胞的转染方法是磷酸钙沉淀，主要是因为最低设备的和试剂所需11,15。替代方法，即电穿孔，脂质体，阳离子脂质和聚合物，可以得到依赖于细胞系更高的转染效率和构造使用。在一般情况下，影响的转染效率的主要因素是质粒DNA的质量和细胞的健康/生存力。当使用纯度最高（的260nm处/ 280nm的吸光度比〜1.8），并积极分裂的细胞的质粒DNA的最佳的结果实现的。细胞因此应该在不超过60被转染- 70％汇合（步骤2.1.2），因为占用外源DNA的能力有关，暴露于介质11中的细胞的表面积。此外，转染（步骤2.1.3）中的培养基中的抗生素夹杂不会由于增加的细胞死亡15宜。 F或磷酸钙沉淀在2X HBS溶液（步2.1.1）的特别精心准备，调整pH（7.05精确），和质粒DNA /钙/剧烈混合（步骤2.1.5），磷酸盐复合物形成正确的高效率转染的关键步骤。通常情况下，通过在24瞬时转染峰表达 - 72小时。 一旦收获细胞，随后的程序必须在4℃下进行以最小化蛋白酶的活性，并且推荐添加蛋白酶抑制剂。细胞均化之后应离心步骤以除去细胞核，因为染色体DNA可以增加溶液的粘度和提高的非特异性结合。因此，同质化通过在等渗缓冲机械手段是首选，一般在（0.3米），蔗糖或（150毫米）的NaCl。在一般情况下，基于蔗糖的缓冲液是已知的，以提高蛋白质稳定性，并减少潜在的非天然蛋白质聚集，但由于为p蛋白质表面上的参照水化，静电蛋白质 - 蛋白质相互作用是有利的。相反地，基于盐缓冲剂影响蛋白质表面上的净电荷电荷的氨基酸侧基，从而具有向更基于疏水相互作用28的偏置。 共IP是最常用的生化测定来评估特别是从天然组织蛋白质 - 蛋白质相互作用。它的主要缺点是为验证为在使用IP和免疫印迹10,16,18高度特异性抗体的要求。因此，重组蛋白质通常具有标记的肽的表位， 例如 ，流感血凝素（YPYDVPDYA）或人cMYC的（EQKLISEEDL），其高亲和力特异性抗体是市售的。如果需要，免疫沉淀抗体可以化学缀合到蛋白A树脂，以避免其在免疫印迹阶段洗脱并检测可能混淆的共沉淀的公关otein 16;实现这一目标，我们已经成功地使用了化学交联剂3,3&#39;-二硫代双（sulfosuccinimidylpropionate）24。至关重要的是，一个合适的洗涤剂包括在IP缓冲液和匀化细胞的不溶性物质通过离心除去非特异性结合（步骤3.1.3）最小化。选择和洗涤剂的浓度是重要的考虑因素:更强洗涤剂和/或更高的浓度将大大降低非特异性结合，但是也可以废除X:Y蛋白质相互作用，而较低的浓度或较温和的清洁剂可允许观察到弱相互作用，但可能导致过度非特异性结合。中间强度洗涤剂是优选的， 例如 ，曲通X-100 0.5 - 1％的浓度。为了进一步减少非特异性结合，中性蛋白质（ 例如，在100微克/毫升牛血清白蛋白），可以包括在IP缓冲液和/或细胞裂解物可以预先cleareð单独用蛋白A树脂前孵化。洗涤的数量应为X进行优化:Y对测试，通常3个10分钟洗涤用的IP缓冲液（步骤3.1.9）。在任何情况下，与非免疫的IgG共IP样品作为免疫沉淀抗体应始终并行处理作为阴性对照（步骤3.1.6）。 化学交联的主要优点是，它通知在蛋白质同源低聚物的化学计量组成。戊二醛是一种常用的交联剂，因为它不需要特殊的设备和它产生相互作用蛋白19,20之间的热和化学稳定的交联。与游离氨基化合物应当从分析缓冲（步骤3.2.1）被省略，因为它们将淬灭的化学反应。戊二醛浓度和反应时间（步骤3.2.4）应为感兴趣的寡聚蛋白复合物进行优化。这种技术，especi的主要缺点对全细胞制剂进行联合时，是化学反应，可以得到缺乏生物学意义人工蛋白聚集体的非特异性。 替代体内 （ 例如 ，荧光共振能量转移，双分子荧光或发光互补）和体外技术（ 例如 ，大小排阻层析，分析超速离心，等温滴定量热法）可用于蛋白质自联合和评估的特征齐聚化学计量29,30。每种方法都有自己的优点和缺点，它可能更适合取决于蛋白质纯化/稳定性和设备/试剂可用特定蛋白质的研究。详细地说，即Y2H，共IP和交联这里描述的三个互补的方法，已在组合被用于对RyR2的同源oligome提供了令人信服的证据- [R形成隔离和活细胞内。

骨骼肌和心肌的收缩是由碱受体介导的肌质网的 Ca 2+释放触发。有三种哺乳动物RyR的同种型具有四个相同的亚基构成的功能信道。每个RyR的亚基由一个大细胞质监管N-末端部分和包含形成高电导钙孔隙跨膜结构域的小C末端部分的。不正常的内部和亚基间的相互作用背后碱受体通道功能障碍，并导致神经肌肉和心脏疾病1。参与碱受体结构特定领域的识别和表征:功能关系，因此对于RyR的病理生理学的理解是至关重要的。 传统的生化蛋白质 - 蛋白质相互作用技术需要的纯化蛋白的显着量，在细菌中经常产生的。这不是在RyR的，非常大的膜p的情况下，可行的rotein由〜5000个氨基酸，而其重组片段不容易适合于细菌表达和纯化。因此，涉及到真核宿主细胞的替代表达系统都需要的哺乳动物整合膜蛋白。我们先前采用Y2H，共IP和交联测定法，以共同表明N末端 ​​四聚是保守的横跨三个哺乳动物RyR的亚型2,3的结构特征。重要的是，我们已发现，致心律失常性心脏疾病相关的单点突变破坏N末端 ​​自联合并以不正常的RyR的信道4的结果。我们还应用这些技术的RyR的细胞质C-末端尾部5以及同源细胞内Ca 2+释放通道的N末端 ​​，所述1,4,5-三磷酸肌醇受体2的低聚研究。 在Y2H测定中，interacti两个在之间蛋白质（X和Y）是由功能性转录因子（GAL4）和报告基因6-9的随后活化的重组测定。两个不同的克隆载体被用于产生与GAL4的两个在物理上可分离的，独立的域两种试验蛋白的融合体:DNA结合结构域（DNA-BD）/ X蛋白融合（诱饵）与激活域（AD）/蛋白质ÿ融合（目标）。在Y2H可用于测试通过产生GAL4的DNA-BD和相同的蛋白的AD融合蛋白是否与自身相互作用。遗传修饰Y2H菌株GAL4和GAL80缺陷（在GAL80蛋白是GAL4的阻抑），以及TRP1和LEU2缺陷（为诱饵和猎物质粒，提供营养选择分别）。在酵母细胞核中，该重组DNA-BD和AD肽被带入物理非常接近，只有通过其融合物"X，以产生一个混合GAL4转录因子:Y INTeraction。这种方法能够快速遗传筛查通过原养型的平行转录激活（HIS3编码所必需的组氨酸生物合成的酶）（对于β半乳糖苷酶（β-GAL） 的LacZ编码）报告基因和显色以检测蛋白质-蛋白质相互作用。在Y2H的主要优点是，它是一种体内测定，其检测甚至弱或瞬态蛋白质-蛋白质相互作用。此外，检测涉及简单的使用增长的选择（在介质缺乏组氨酸）或比色的（β-GAL）测定，无需为诱饵和靶蛋白的纯化或特异性抗体的产生。此外，Y2H可用于筛选随机未知克隆（cDNA文库克隆融合至GAL4 AD）为一诱饵蛋白的新颖的结合配偶，也给人以库蛋白的cDNA的直接访问的集合。 为了延长Y2H观察，独立工作耳鼻喉科生化技术都可以使用。共IP和交联测定法用免疫印迹结合是用于检测在复杂样品混合物蛋白协会方法， 例如 ，全细胞裂解物10。它们的主要优点是它们对蛋白质 - 蛋白质相互作用报告从天然组织，不像需要使用重组蛋白质的其它方法。重组蛋白质也可使用，通常在哺乳动物细胞系，在那里它们可能有它们的正确构象和翻译后修饰，以及亚细胞定位表达。然而，由于共同的IP和交联是在体外测定利用匀浆细胞，有必要确认是否两个蛋白伙伴是在完整细胞11共定位。我们经常使用哺乳动物HEK293细胞转染使用磷酸钙沉淀法2瞬时表达哺乳动物完整的膜和胞质蛋白-4,12-14，在这里详细描述。这是为了有效地提供在细胞内的质粒DNA的一种廉价的方式，但它是依赖于所使用的特定细胞系和细胞融合，以及质粒DNA 11,15的纯度。 共同的IP测定涉及的非变性条件，使推定的相互作用配偶10,16的共同纯化下从细胞裂解物感兴趣的天然或重组蛋白的分离。它需要使用两个独立的抗体，对蛋白质的X隔离免疫沉淀抗体，以及用于检测蛋白伴侣Y的免疫印迹的抗体可用于与两个不同的表位，以测试通过产生两种不同的融合蛋白是否与自身进行交互标签（ 如 ，HA和cMYC的）。在免疫沉淀抗体通过其Fc区结合的蛋白A（或蛋白G，取决于其中抗体引发的动物种类），其缀合到琼脂糖（或琼脂糖）树脂。在用细胞裂解产物，即均化细胞的洗涤剂可溶性级分温育蛋白A树脂:蛋白质X由抗体沉淀。蛋白免疫复合物洗脱含有SDS的缓冲液，随后通过SDS-PAGE分析和使用抗体来检测蛋白的存在ý17的免疫印迹。共IP应用洗涤剂可溶蛋白进行，以避免过多的非特异性结合。 y对10,16,18:用洗涤剂和它的浓度的选择，以及洗涤次数，应为每个X进行优化。 交联是用来确定所述寡聚蛋白复合物的化学计量。它是基于化学反应来创建相邻相互作用原聚体之间形成共价键，因此，它使SDS-PAGE分离时的蛋白的寡聚状态保存。有许多交联reage各种长度和化学上的蛋白质，通常伯胺，羧酸或硫醇基团靶向不同反应性基团的NTS。在这里，我们描述了使用戊二醛（OHC（CH 2）3 CHO），一个同型双功能交联剂与在任一端是与存在于赖氨酸残基19,20游离氨基反应两个醛基。交联之后是在产生加合物的形成以浓度或时间依赖性。戊二醛反应停止用肼（H 2 NNH 2）和蛋白质样品，然后通过SDS-PAGE分析和免疫印迹17，以评估其低聚状态。我们应该注意到，交联不诱导低聚而仅仅创建预先存在的蛋白质复合体之间的稳定桥。在进行交联实验时的重要考虑因素包括交联剂，其浓度和反应时间19,20的选择。

<table><tbody><tr><td>&lt;strong&gt;PART 1 yeast two-hybrid&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plate incubator</td><td>Hereaus</td><td>B6120</td><td>Used at 30 &amp;deg;C</td></tr><tr><td>Orbital shaker incubator</td><td>New Brunswick Scientific</td><td>INNOVA 4300</td><td>Used at 30 &amp;deg;C with shaking at 230 - 250 rpm</td></tr><tr><td>Spectrophotometer</td><td>Perkin Elmer</td><td>Lambda Bio+</td><td>To measure OD&lt;sub&gt;600&lt;/sub&gt; of yeast culture; to measure Absorbance at 420 nm in liquid b-Gal assay</td></tr><tr><td>Matchmaker Two-Hybrid System 2 Kit</td><td>Takara Clontech</td><td>K1604-1</td><td>Contains bait vector pGBKT7, prey vector pACT2&amp;nbsp; and yeast strain Y190&amp;nbsp;</td></tr><tr><td>Yeast Nitrogen Base</td><td>Sigma-Aldrich</td><td>Y0626</td><td>To prepare minimal SD growh medium&amp;nbsp;</td></tr><tr><td>Dropout supplement lacking leucine and tryptophan</td><td>Sigma-Aldrich</td><td>Y0750</td><td>To prepare minimal SD growh medium&amp;nbsp;</td></tr><tr><td>Dropout supplement lacking leucine, tryptophan, histidine</td><td>Sigma-Aldrich</td><td>Y2001</td><td>To prepare minimal SD growh medium&amp;nbsp;</td></tr><tr><td>Herring testes carrier DNA&amp;nbsp;</td><td>Takara Clontech</td><td>630440</td><td>For yeast transformation</td></tr><tr><td>Whatman filter paper Grade 5&amp;nbsp;</td><td>Sigma-Aldrich</td><td>WHA1005070</td><td>for use in colony-lift filter paper b-Gal assay</td></tr><tr><td>X-Gal (5-bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside)</td><td>Sigma-Aldrich</td><td>B4252</td><td>for use in colony-lift filter paper b-Gal assay</td></tr><tr><td>ONPG (o-nitrophenyl-&amp;beta;-D-galactopyranoside)</td><td>Sigma-Aldrich</td><td>N1127</td><td>for use in liquid b-Gal assay</td></tr><tr><td>&lt;strong&gt;PART 2. Protein expression in a mammalian cell line&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HEK293 cell line</td><td>ATCC</td><td>ATCC&amp;reg;&amp;nbsp;CRL-1573&amp;trade;</td><td></td></tr><tr><td>Humidified incubator (5% CO&lt;sub&gt;2&lt;/sub&gt;, 37 &amp;deg;C)</td><td>SANYO</td><td>MCO-18AIC</td><td>To culture mammalian cells</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle&#039;s medium)</td><td>Invitrogen (ThermoFisher)</td><td>41966</td><td>To prepare growth medium for mammalian cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen (ThermoFisher)</td><td>10500</td><td>To prepare growth medium for mammalian cells</td></tr><tr><td>Glass beads</td><td>Sigma-Aldrich</td><td>G8772</td><td>To homogenise cells</td></tr><tr><td>Needle 23 G (0.6 x 30 mm)</td><td>BD Microlance</td><td>300700</td><td>To homogenise cells</td></tr><tr><td>Protease inhibitor cocktail (Complete)&amp;nbsp;</td><td>Roche</td><td>11873508001</td><td>To prevent proteolysis</td></tr><tr><td>&lt;strong&gt;PART 3. In vitro biochemical methods&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell (SDS-PAGE system)</td><td>Bio-Rad</td><td>1658000EDU&amp;nbsp;</td><td>For polyacrylamide gel electrophoresis</td></tr><tr><td>Trans-Blot SD (Semi-dry transfer system)</td><td>Bio-Rad</td><td>1703940</td><td>For electrophoretic transfer</td></tr><tr><td>Compact X-ray film processor</td><td>Xograph</td><td>X4</td><td>For use in immunoblotting</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882</td><td>For use in chemical cross-linking</td></tr><tr><td>Protein-A sepharose beads&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>17-5280-01&amp;nbsp;</td><td>For use in co-IP assay</td></tr><tr><td>Anti-HA (Y-11 rabbit polyclonal IgG)</td><td>Santa Cruz Biotechnology</td><td>sc-805</td><td>For use in co-IP assay</td></tr><tr><td>Non-immune rabbit IgG</td><td>Santa Cruz Biotechnology&amp;nbsp;</td><td>sc-2027</td><td>For use in co-IP assay</td></tr><tr><td>Anti-cMyc (9E10 mouse monoclonal IgG)</td><td>Santa Cruz Biotechnology&amp;nbsp;</td><td>sc-40</td><td>For use in immunoblotting</td></tr><tr><td>Anti-HA (16B12 mouse monoclonal IgG)</td><td>Covance</td><td>MMS-101P</td><td>For use in immunoblotting</td></tr><tr><td>Anti-mouse IgG-HRP</td><td>Santa Cruz Biotechnology</td><td>sc-2005</td><td>For use in immunoblotting</td></tr><tr><td>Hyperfilm ECL</td><td>GE Healthcare Life Sciences</td><td>28906836</td><td>For use in immunoblotting</td></tr><tr><td>ECL Western Blotting Substrate</td><td>Pierce (ThermoFisher)</td><td>32106</td><td>For use in immunoblotting</td></tr></tbody></table>

genetic and biochemical approaches for in vivo and in vitro assessment of protein oligomerization: the ryanodine receptor case study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

在Y2H系统中，诱饵:猎物相互作用最初是由在介质酵母生长选择缺乏（色氨酸，亮氨酸和）组氨酸测试并随后通过β-Gal的酶活性测定法（ 图1）进行评估。酵母培养基中缺乏组氨酸的培养具有取决于诱饵的强度增长缓慢:猎物蛋白质相互作用。将β-Gal的测定在酵母中进行（在培养基中培养仅缺少色氨酸和亮氨酸）或生长在固体支持物（琼脂平板），或在液体培养，后者屈服定量结果。我们已经成功地使用了Y2H识别RyR2的内域的域相互作用以及新型蛋白伙伴2-4,12,21,22。例如，我们筛选一系列重叠构建跨越RyR2的肽序列的相互作用的整个长度具有N-末端片段的（AD4L:RyR2的残基1 - 906与GAL4 AD融合）3菌落升降机滤纸β-Gal的测定仅产生用于BT4L生动蓝色的酵母菌落:AD4L对（ 图2A），表明AD4L相互作用与本身，即BT4L构造（RyR2的残基1 -稠合与GAL4 906 DNA-BD）。为BT8检测淡蓝色菌落:AD4L对暗示与极端C-末端结构域（BT8）仲弱关联，而与任何其它构建酵母共转化保持白色，因此负为诱饵:猎物蛋白质相互作用。定量的结果，由液体β-Gal的测定法（ 图2B），获得指示健壮BT4L:AD4L相互作用相当于强度对p53蛋白（PVA3）和SV40大T抗原（PTD1）之间的已知的关联，而BT8:AD4L相互作用被大大减弱（&lt;10％，与对照相比）。 我们定期开展联合实验的IP（ 图3）的F在哺乳动物细胞系（HEK293）ollowing瞬时表达，作为一个独立的生物化学测定，以加强Y2H发现2-4,12-14,21-24。为了验证RyR2的N端自身相互作用，编码RyR2的残留两个独立的质粒1 - 906标记无论是cMYC的或HA肽表（BT4L和AD​​4L，分别），采用磷酸钙沉淀法在HEK293细胞共转染3。匀浆细胞后核子馏分与洗涤剂CHAPS溶解，并通过离心除去不溶性物质，以产生细胞裂解物。细胞裂解物，与还原剂二硫苏糖醇处理，然后用抗体HA和蛋白A琼脂糖珠培养免疫沉淀HA标记AD4L。共IP的样品，用含有SDS的缓冲液洗脱，被装在两个独立的SDS-PAGE凝胶（1/10和9/10 个分割）和分析用抗体HA和抗体cMYC的河免疫印迹espectively。 AD4L成功直接IP（〜100 kDa的）由抗体的HA通过免疫印迹证实，但在使用非免疫兔IgG（ 图4A）的阴性对照。重要的是，cMYC的标记BT4L（〜100 kDa）的回收仅在抗体的HA的IP，而不是在不存在免疫沉淀AD4L（ 图4B）的阴性对照。 Y2H和共同的IP试验提供一致的证据为RyR2的N末端自相互作用，但他们并没有通知上的N-末端结构域的寡聚化状态，即是否它形成仅二聚物或更高的复合物。应当注意的是，复合物将分离并仅包括亚基将通过SDS-PAGE因为SDS-的和热诱导蛋白质变性废除蛋白 - 蛋白相互作用来检测。为了克服这个问题，我们用交联（ 图5），该化学和稳定地星相预先存在proteiÑ ​​低聚物，其分子量然后可以通过SDS-PAGE尺寸分离2-5进行检查。例如，表达cMYC的-BT4L，与还原剂二硫苏糖醇处理的HEK293细胞匀浆，用戊二醛反应，通过使用抗体cMYC的 （ 图6）的SDS-PAGE和免疫印迹分析。除了 ​​单体（〜100 kDa的），千道尔顿〜400高的分子量的蛋白条带在一个时间依赖性检测，指示的RyR2 N末端 ​​四聚体形成3。值得注意的是，四聚体是主要的低聚物种，以最少的二聚体和三聚体观察到条带。 15％梯度SDS-PAGE凝胶3 -来确定其表观分子质量，该BT4L低聚物是通过4分离。我们生产使用蛋白质标准与范围30的分子质量/凝胶阻滞标准曲线 - 460 kDa的，并且我们计算该低聚物为358 kDa的15（N = 4）。这种表观分子量是用设置在一个BT4L四聚体一致的闭合循环的方式而不是线性形式，如从天然RyR2的通道内的四个亚基的结构预期。 <img alt="图1" src="/files/ftp_upload/54271/54271fig1.jpg" /> 图1. Y2H流程图。酵母，共转化诱饵和目标质粒，选择用于中缺乏组氨酸的生长和/或测定β-Gal的酶活性。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54271/54271fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54271/54271fig2.jpg" /> 图2. Y2H:建议的RyR2 N末端 ​​域自相互作用。（A） 的示意图描绘了（诱饵）系列在Y2H系统测试人的RyR2重叠蛋白片段<html>与RyR2的N端AD4L（猎物）结构的相互作用。通过菌落升降滤纸β-Gal的测定法获得的定性结果被表示在"+"的倍数或" - "为负相互作用归对阳性对照（B）的定量液体β-Gal的测定（PVA3编码GAL4的DNA-BD。融合p53蛋白; PTD1编码与SV40大T抗原GAL4 AD融合）。从3修改。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54271/54271fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54271/54271fig3.jpg" /> 图3. 共同的IP流程图。哺乳动物细胞，共转染质粒的X和Y，均化并洗涤剂溶解，以产生在共IP测定中使用的细胞裂解物，随后通过SDS-PAGE和免疫印迹。F ="htt​​ps://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54271/54271fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54271/54271fig4.jpg" /> 图4 共同的IP表示RyR2的N末端 ​​域自相互作用在哺乳动物细胞中。HEK293细胞进行共转染cMYC的标记（BT4L）和HA-标记（AD4L）RyR2的N-末端结构域的瞬时共表达（残留物1 - 906）。 AD4L用抗体的HA免疫沉淀从CHAPS溶解的和二硫苏糖醇处理的HEK293裂解物，而作为阴性对照，共IP测定法用非免疫兔IgG进行。免疫沉淀的蛋白质在85℃下加热5分钟，并在20mA解析为通过装载有1/10或9/10 个 IP样品分开6％SDS-PAGE凝胶3小时。在80伏以下的蛋白转移2小时到polyviny乙叉二氟乙烯膜，免疫印迹分析进行了使用（1:1000稀释）抗体的HA（A）或抗体cMYC的 （B）中 ，分别，随后辣根过氧化物酶缀合的抗小鼠IgG（1:10,000稀释）和增强的化学发光检测（1分钟曝光）。细胞裂解物的等分试样，IP样品中处理的体积的1/50 个 ，也包括以用作分子量标准。从3修改。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54271/54271fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54271/54271fig5.jpg" /> 图5. 交联流程图。哺乳动物细胞，用质粒点¯x转，均化并进行反应，在交联测定法戊二醛，接着进行SDS-PA的GE和免疫印迹。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54271/54271fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54271/54271fig6.jpg" /> 图6. 交联表示RyR2的N末端 ​​域形成四聚体 HEK293细胞转染cMYC的标记（BT4L）RyR2的N端结构域的瞬时表达（残基1 - 906）。细胞匀浆，用还原剂二硫苏糖醇处理后，用戊二醛在指定的时间点温育。样品在85℃下进行5分钟的加热和在20mA通过SDS-PAGE（6％凝胶）分辨3小时。以下在80伏的蛋白质转印2小时到聚偏二氟乙烯膜，免疫印迹分析进行了使用（1:1000稀释）抗体cMYC的 ，其次是辣根过氧化物酶缀合的抗小鼠IgG（1:1:10,000稀释）和增强的化学发光检测（1分钟曝光）。单体（M:〜100 kDa的）和四聚体（T）的表格由箭头指示。从3修改。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54271/54271fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study at JoVE.com

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估:兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

genetic-biochemical-approaches-for-vivo-vitro-assessment-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

11.6K 次观看.  Cardiff University School of Medicine, College of Biomedical and Life Sciences. Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

视频: Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-&#946; peptide variants with their respective intrinsic aggregation propensities.

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-&#946; peptide.

蛋白质聚集对酵母细胞氧化应激的影响评价

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

科研

生物化学

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

龙苷受体 n 端域的晶体结构

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

管式膜网络在心肌细胞的心房和心室分析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

生物工程

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

使用慢病毒介导的CRISPR / Cas9基因编辑开发敲除肌肉细胞系

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, ...

遗传学

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

高含量成像分析研究。

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

化学

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

生物学

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.

We describe a protocol to induce phase transition of TAR DNA-binding protein 43 (TDP-43) by light in the spinal motor neurons using zebrafish as a model.

TDP-43在斑马鱼幼虫脊髓运动神经元中的光遗传学相变

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between ...

神经科学

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

内源性表达人RYR1变异体的功能表征

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

医学

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes following physical activity, stress, or catecholamine challenge, which can deteriorate into potentially fatal ventricular fibrillation. The mouse heart is a widespread species for modeling inherited cardiac arrhythmic diseases, including CPVT. Simultaneous optical mapping of transmembrane potential (Vm) and calcium transients (CaT) from Langendorff-perfused mouse hearts has the potential to elucidate mechanisms underlying arrhythmogenesis. Compared with the cellular level investigation, the optical mapping technique can test some electrophysiological parameters, such as the determination of activation, conduction velocity, action potential duration, and CaT duration. This paper presents the instrumentation setup and experimental procedure for high-throughput optical mapping of CaT and Vm in murine wild-type and heterozygous RyR2-R2474S/+ hearts, combined with programmed electrical pacing before and during the isoproterenol challenge. This approach has demonstrated a feasible and reliable method for mechanistically studying CPVT disease in an ex vivo mouse heart preparation.

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

This protocol introduces dual-dye optical mapping of mouse hearts obtained from wild-type and knock-in animals affected by catecholaminergic polymorphic ventricular tachycardia, including electrophysiological measurements of transmembrane voltage and intracellular Ca2+ transients with high temporal and spatial resolution.

儿茶酚胺能多形性室性心动过速的 RyR2R2474S 敲入小鼠心脏的双染料光学映射

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes ...

遗传和生物化学方法

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