这项工作是由国家健康授予GM086822，希望心脏研究所的基础科学奖，兆焦耳默多克慈善信托基金科学学院研究计划研究院资助。选择电泳试剂和图像分析软件Bio - Rad公司提供的慷慨。

1。制备细胞细胞质中含有功能的GR <ol><li>技术说明：所有的缓冲区应冷藏，此协议，包括离心和孵化，每一步应在冰上或在4 ° C，除非另有说明。低温是必不可少的，以防止GR和蛋白质复合物的降解。 </li><li>使用冷冻离心机，取得了〜1-5毫升颗粒细胞功能的GR表达了高浓度。在遗传资源丰富的细胞来源的例子包括：小鼠成纤维L929细胞，它表达的内源性遗传资源的高浓度，并昆虫重组GR杆状病毒（如p2Bac 经理杆状病毒12）已被感染的细胞。大约15-20毫升包装细胞获得每昆虫杆状病毒细胞培养升。细胞应呈几何级数增长，前收获。在5000 × g离心5分钟的细胞悬液。 </li><li>汉克斯“缓冲盐溶液15毫升，冲洗细胞沉淀三次。清洗，轻轻悬浮5分钟，随后在5000离心沉淀× G。最后一次洗涤，彻底去除上清。 </li><li>准备7.5毫升含HEM的缓冲液（10 mM的HEPES，1毫米EDTA，20毫米钼酸钠，pH值7.4），1毫米苯甲基磺酰氟（PMSF），和3完成迷你蛋白酶抑制剂的地面片的缓冲一个同质化。 PMSF和完整的小片在随后的步骤防止发生水解。完整的迷你片，可以很容易地地面到细粉用迫击炮和杵。添加1.5卷同质缓冲细胞沉淀。 </li><li>破裂Dounce同质化（50杆）或冻结/解冻（3次）技术的细胞。 Dounce同质化可能维持改善内源性的GR•HSP90蛋白复合物，并更快地完成。冻结/解冻提供研究人员一个机会，暂停在这一步的协议，并继续在稍后的时间。 Dounce同质化应与冰桶匀浆，以防止蛋白质的降解。冻结/解冻，可完成冻结的离心管中，含有颗粒同质化缓冲液的细胞用液氮，干冰或-20 ° C孵化，在30℃水浴解冻。 </li><li>通过超速离心分离从破裂的细胞颗粒物质的GR含有细胞质。在100,000 × g匀浆转移耐用的超速离心机管，对群众的平衡管和离心30分钟。 </li><li>取出500μL分装在长期储存的1.5毫升离心管，在-20细胞质（上清液）和存储° C。要保留的最大的功能GR活动，闪存冻结30秒甲醇干冰浴前存储在液态氮或孵化分装。一定要小心，以避免皮肤直接接触冷却剂。 </li></ol> 2。从细胞细胞质遗传免疫吸附<ol><li>技术说明：图2提供了这个协议的步骤2-5的示意图。 </li><li>准备一个包含immunoadsorb在第1步准备的受体对GR提出一个单克隆免疫球蛋白（IgG）抗体的免疫吸附混合物。在1.5 ml离心管中，结合解冻的GR - 100μL含有细胞质中，200μLTEGM缓冲区（8毫米的工商业污水附加费，4毫米EDTA，50毫米的NaCl，20毫米的钼，10％（V / V）甘油，pH值7.6） 100μL20％的蛋白质A -琼脂糖凝胶（PAS）浆（V / V，准备在TEGM缓冲区），5微克抗- GR的单克隆抗体（如BuGR2）。钼是包括在TEGM缓冲区，以帮助稳定GR - HSP90 heterocomplex，这可能是有用的，以测定的内源性heterocomplex类固醇结合活动 13 。 <ol><li>反GR抗体纯化蛋白上市。另外，它可能会取得反GR的含有IgG的腹水免疫吸附混合物中加入10μL。腹水是由小鼠注射抗- GR杂交瘤细胞（如FiGR杂交瘤细胞14），并获得腹水和在这些实验中使用的是近似的抗体浓度为0.5μg/μL，Bradford法测定。 </li><li>准备一个阴性对照样品，使用GR - 含有细胞质中，TEGM缓冲区，和首席助理秘书长（如上所述），一个IgG和5微克不承认的GR，HSP90，或其他分子伴侣蛋白（简称为“非免疫”抗体）。 </li><li>由于比较大的PAS珠子大小，使用切割时的P - 200吸管尖转移的20％浆PAS样品管。切吸管提示准备切片2-3毫米市售的P - 200提示结束，从而增加了吸管尖光圈。 </li></ol></li><li>将样品在垂直旋转的轮子和2小时为一个不断旋转的最低孵化。样品可能长达8小时的孵育，而不失去活性。 </li><li>在免疫吸附孵化结束，去除未结合的蛋白质抗体考绩颗粒（“immunopellet”）离心。从immunopellets分开使用一个冷冻离心机1分钟编程10,000 × G离心的上清液，然后用1 ml TEGM缓冲区和涡流清洗两次。经过离心和洗涤之间，弃上清，谨慎，不打扰颗粒。 <ol><li>删除所有在第二洗的结论上清。为了确保没有颗粒不经意地吸气，用压接的P - 200吸管尖，最终上清愿望。轧花吸管提示准备压扁市售的P - 200的提示使用镊子。 </li></ol></li></ol> 3。从遗传资源immunopellet内源性HSP90分解（“盐剥离”） <ol><li>要洗immunopellet准备在第2步，添加355μL甘醇缓冲液（TEGM没有钼酸钠缓冲液）和45μL5 M氯化钠（NaCl浓度最终= 0.5米）。 </li><li>将样品在垂直旋转的轮子和不断旋转的1.5小时的孵化。样品可以孵育长达2个小时，但是，较长的孵化，可能会导致减少蛋白质的恢复和功能活性较低。 </li><li>取出离心的immunopellet未结合的蛋白质。 1分钟，在10000 × G.旋转样品洗一次1毫升甘醇缓冲​​区，并一度以1毫升10 mM的HEPES缓冲液，pH值7.4。 </li><li>删除所有在第二洗的结论上清，小心，不要去打扰immunopellet，使用一个卷曲的P - 200吸管尖。 </li></ol> 4。重组的GR•HSP90 heterocomplex使用分子伴侣，辅助因子，与ATP <ol><li>技术说明：无​​数的实验条件，可以测试到准备下面的重组混合物，包括额外的蛋白质，辅助因子，激活剂，或利益抑制剂。重组混合物的总体积应&lt;120μL。 </li><li>每个盐剥离immunopellet准备在第3步，添加一个重建的混合物，含有50μL分子伴侣源和5μL一个ATP生成系统（10 mM的HEPES 50 mM的ATP，磷酸肌酸250毫米，20毫米醋酸镁100 U / ml的肌酸磷酸激酶，pH值7.4）。 <ol><li>重整反应，可能会增加补充重组混合物50μL港币缓冲液（10 mM的HEPES，氯化钾100毫米，5毫米二硫苏糖醇（DTT），pH值7.4）和ATP生成系统的一个额外的5μL。如果观察充足的重组和类固醇具有约束力，这一步可以省略。氯化钾增加HSP70的ATP酶活性及数码地面电视保护半胱氨酸含蛋白质氧化 9 。 </li><li>一个推荐的分子伴侣来源是市售的兔网织红细胞裂解物。个别分子伴侣从网织红细胞裂解液纯化或重组表达（如15微克HSP90，HSP70 15微克，0.6微克合，P23 6微克和0.125微克港元缓冲区hsp40），也可使用。 </li></ol></li><li>在30℃水浴整整20分钟中孵育样品。弗里克管，以每1-2分钟，轻轻扰乱颗粒混合重组解决方案。 </li><li> 1分钟，加入1毫升TEGM缓冲区，旋涡，在10,000和离心机× G。小心，不要去打扰颗粒去除上清液并丢弃。共有3个洗重复。在缔结最终使用的P - 200压接吸管尖洗，彻底清除所有上清。 </li></ol> 5。重组蛋白复合物与配体结合活性分析<ol><li>在重组immunopellet中的蛋白质可以识别常规聚丙烯酰胺凝胶电泳（SDS - PAGE），微流控电泳（如Bio - Rad公司的Experion），和免疫印迹（ 图 3 ）。技术说明：优秀的协议， 描述 15 SDS - PAGE电泳和免疫印迹 16目前，其他调查员的礼貌。 <ol><li>添加50μL的SDS - PAGE样品缓冲液含有β-巯基乙醇和涡。特定的样品缓冲液配方有所不同。被聘用的电泳系统的制造商建议的基础上，选择之一。 </li><li>孵育5分钟，在沸水浴或100 ° C电子加热块。 </li><li>在10,000 × g离心1分钟。 immunoadsorbing抗体，GR蛋白复合物遗传资源将脱离彼此释放到样品缓冲液上清中。 </li><li>将上清转移至一个新的离心管，用吸管尖的P - 200压接尖。弃去沉淀。正准备用市售的SDS - PAGE或微流控电泳系统分析样品。完成两种技术following制造商的指示。 </li><li> Western blot分析以下的SDS - PAGE可为最终鉴定immunoadsorbed遗传和分子伴侣的GR复合重组孵化。使用对GR（BuGR2），HSP90（AC88）提出的单克隆抗体，HSP70（N27F3 4）和合作伴侣蛋白检测GR•HSP90蛋白复合物的主要成分。 </li></ol></li><li> •HSP90蛋白复合物，可以通过化验使用类固醇的结合活性[3H] 类固醇（图 4）确定遗传资源的重组后，盐剥离的GR功能激活。对于该协议的其余部分，遵循必要的预防措施，安全处理氚的样品和废物。 <ol><li>重组后immunopellet，添加47.5μLHEM的缓冲区2微米和2.5μL[3H]地塞米松（最后的类固醇浓度= 100纳米） 。 </li><li>轻轻地混合沉淀小心，尽量减少流离失所的样品量的离心管的墙。 </li><li>在冰上孵育过夜。它是没有必要的旋转或在此孵化混合样品管的。 </li><li>添加1毫升TEGM缓冲液，轻轻混匀，在10,000和离心机× G为1分钟。弃上清，被铭记，它包含未绑定的[3 H]类固醇。重复共3 TEGM缓冲液洗。 </li><li>在缔结最终使用的P - 200压接吸管尖洗，彻底清除所有上清。颗粒包含的GR，HSP90和其他分子伴侣复合物的GR，[H]类固醇受体特异性结合。非特异性结合，可以计算，包括1000倍的超额非氚在地塞米松孵育过夜混合物。作为替代阴性对照，反GR immunoadsorbing抗体可能会被替换在步骤2.2中添加非GR - immunoadsorbing（NI）的抗体。 </li><li>挂起200μLTEGM缓冲区，并转移至10 ml闪烁瓶使用的P - 200吸管尖切洗immunopellets。 </li><li>加入4.8毫升闪烁鸡尾酒瓶和涡。 </li><li> [3H]类固醇结合到重组后的GR金额•HSP90蛋白复合物，可以使用液体闪烁光谱法测定。 </li></ol></li></ol> 6。代表性的成果： 代表SDS - PAGE和免疫印迹数据列在图3。内源性的GR•HSP90 heterocomplex immunoadsorbed使用反GR抗体和合作与GR immunoadsorb考马斯染色（图3A）显示的个别蛋白质从细胞的细胞质。蛋白质在重组实验的任何步骤的immunopellet任何组件的身份确认，可经SDS - PAGE计算分子量和使用提出对感兴趣的蛋白质的单克隆抗体免疫印迹。 冲调的GR•HSP90 heterocomplexes是使用的Experion微流体电泳数据（图3C和3D）和免疫印迹分析（图3B）。确认的GR免疫吸附特异性与非免疫抗体（图3C，NI IgG抗体车道）取代immunoadsorbing抗体抗- GR。即使过量使用非免疫抗体免疫沉淀，没有遗传或分子伴侣（参见图3c，GR，HSP90，HSP70蛋白带目前所观察到的带缺乏反GR免疫吸附车道NI IgG的车道）。同样，它可能证实了HSP90的分离和其他伴侣蛋白，盐剥离的剥离和重组immunopellets克免疫印迹分析（图3B）。考马斯染色和免疫印迹immunoadsorbing抗体的重链（HC）的检测，它是为了确认平等的大小immunopellets在每个泳道显示。 immunoadsorbed的GR结合活性的类固醇可以检测所有的测试条件，和有代表性的类固醇数据绑定在图4。内源性的GR•HSP90 heterocomplexes immunoadsorbed，盐剥离，重组。使用这两种网织红细胞裂解物或纯化的蛋白，重组的GR结合活性的类固醇•HSP90 heterocomplex通常返回的内源性GR•HSP90结合活性水平的75-100％。电泳数据相似，可能是一个非免疫抗体（NI）的反GR抗体（一）地方，以服务为阴性对照，并作为衡量非特异性[3H]类固醇具有约束力。 <img src="/files/ftp_upload/3059/3059fig1.jpg" alt="图1"/> 图1。GR•HSP90 heterocomplex大会和遗传资源类固醇结合裂开放模型。 hsp90/hsp70-based伴​​侣机械转换GR配体结合域从一个FOLDED的构象在类固醇结合裂是封闭的，而不是访问激素可以访问（杂环类固醇图标所示）一个开放的裂构象。伴侣机器预装在细胞和纯化HSP90，HSP70时，会自发形成，合溶液混合（反应1）。合包含34氨基酸tetratricopeptide重复（TPR）域（作为一个黑暗的新月所示），是后来在一个TPR域包含immunophilin蛋白heterocomplex成熟过程中所取代。机械打开类固醇结合的ATP和K +依赖性裂，后合和最hsp70和hsp40最终离开复杂的（图标为虚线所示）。机械，HSP70与GR相互作用之前HSP90和HSP90的受体素数的和随后裂开放。实验中，类固醇具有约束力和GR裂开放增加，如果HSP90和HSP70同时存在。 heterocomplex是稳定的HSP90合作伴侣P23，保持在一个ATP约束构型HSP90的条目。一个超高分子量immunophilin（IMM）合退出后，可以绑定HSP90，形成最终heterocomplex的，因为它是从细胞恢复。改编自普拉特和Toft 1。 <img src="/files/ftp_upload/3059/3059fig2.jpg" alt="图2"/> 图（2）本协议的步骤2-5的示意图。 GR是immunoadsorbed使用绑定的蛋白A琼脂糖凝胶颗粒的GR（FiGR）提出的一种单克隆抗体，对细胞的细胞质。 HSP90和其他伴侣蛋白存在于内源性的GR•HSP90 heterocomplex与GR合作immunoadsorbed，和GR是能够绑定到类固醇。 HSP90是脱离使用温和的盐处理盐剥离（STR），简称GR。类固醇具有约束力的盐剥离的GR裂闭合，使得它无法绑定到类固醇。中级GR•HSP90 heterocomplexes含有HSP70，hsp40，和跳，这是能够约束力的类固醇，可以重组，孵化与纯化蛋白质或网织红细胞裂解液（Retic裂解液）和辅助因子的盐剥离的GR（侦察）。类固醇的结合活性和immunopellet蛋白质含量每一步可以过夜[H]的类固醇孵化和免疫印迹分别确定。 <img src="/files/ftp_upload/3059/3059fig3.jpg" alt="图3"/> 图3。immunoadsorbed复合物电泳的蛋白质分析。一，重组的遗传资源的SDS - PAGE•HSP90 heterocomplex影像使用传统的电泳系统electrotransfer和考马斯染色。此外GR，HSP90和HSP70，重链（HC）和轻链抗体（立法会）的可视化。分子量标记（kDa的表达）的迁移，是显示在左侧。 B，含有盐剥克（STR）和重组GR•HSP90 heterocomplex（侦察）immunopellets免疫印迹。 ，重组遗传资源的虚拟凝胶•HSP90 heterocomplex使用的Experion微流体电泳系统生成的。包括两个不同浓度的一个非免疫（NI IgG抗体），免疫（抗- GR）immunoadsorbing抗体。 NI的IgG为阴性对照。研发，电泳重组的GR•HSP90的Experion微流体电泳显示面板B.微流控电泳的优点，包括较小的样本量的要求第三线的样品获得的heterocomplex，降低液体处理和运行后清理，提高了量化，并大幅较短的样品运行。 <img src="/files/ftp_upload/3059/3059fig4.jpg" alt="图4"/> 图4。类固醇结合活性，免疫吸附内源性的GR（Enodg），盐剥离的GR（STR）immunopellets和重组的GR•HSP90 heterocomplex（侦察）。此外，immunoadsorbing单克隆抗- GR immunoadsorbing抗体（一）GR，每个条件的阴性对照样品，使用非免疫球蛋白（NI）的准备。

<ol>
	<li>Pratt, W. B., Toft, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+signaling+protein+function+and+trafficking+by+the+hsp90/hsp70-based+chaperone+machinery.">Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery.</a> Exp. Biol. Med. 228, 111-133 (2003).</li><li>Murphy, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucocorticoid+receptor+steroid+binding+and+trafficking+by+the+hsp90/hsp70-based+chaperone+machinery:+implications+for+clinical+intervention.">Regulation of glucocorticoid receptor steroid binding and trafficking by the hsp90/hsp70-based chaperone machinery: implications for clinical intervention.</a> Leukemia. 19, 710-712 (2005).</li><li>Pratt, W. B., Morishima, Y., Osawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp90+chaperone+machinery+regulates+signaling+by+modulating+ligand+binding+clefts.">The Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts.</a> J. Biol. Chem. 283, 22885-22889 (2008).</li><li>Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+steroid+receptor&#149;hsp90+heterocomplex+assembly+system+of+rabbit+reticulocyte+lysate.">Reconstitution of the steroid receptor&#149;hsp90 heterocomplex assembly system of rabbit reticulocyte lysate.</a> J. Biol. Chem. 271, 12833-12839 (1996).</li><li>Morishima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp+organizer+protein+hop+enhances+the+rate+of+but+is+not+essential+for+glucocorticoid+receptor+folding+by+the+multiprotein+Hsp90-based+chaperone+system.">The Hsp organizer protein hop enhances the rate of but is not essential for glucocorticoid receptor folding by the multiprotein Hsp90-based chaperone system.</a> J. Biol. Chem. 275, 6894-6900 (2000).</li><li>Murphy, P. J. M., Kanelakis, K. C., Galigniana, M. D., Morishima, Y., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry,+abundance,+and+functional+significance+of+the+hsp90/hsp70-based+multiprotein+chaperone+machinery+in+reticulocyte+lysate.">Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate.</a> J. Biol. Chem. 276, 30092-30098 (2001).</li><li>Dittmar, K. D., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+the+glucocorticoid+receptor+by+the+reconstituted+Hsp90-based+chaperone+machinery.+The+initial+hsp90.p60.hsp70-dependent+step+is+sufficient+for+creating+the+steroid+binding+conformation.">Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70-dependent step is sufficient for creating the steroid binding conformation.</a> J. Biol. Chem. 272, 13047-13054 (1997).</li><li>Kanelakis, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+the+hsp70-binding+protein+BAG-1+on+glucocorticoid+receptor+folding+by+the+hsp90-based+chaperone+machinery.">Differential effects of the hsp70-binding protein BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery.</a> J. Biol. Chem. 274, 34134-34140 (1999).</li><li>Morishima, Y., Kanelakis, K. C., Murphy, P. J. M., Shewach, D. S., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+iterative+ratcheting+of+receptor-bound+hsp70+between+its+ATP+and+ADP+conformations+during+assembly+of+glucocorticoid+receptor.hsp90+heterocomplexes.">Evidence for iterative ratcheting of receptor-bound hsp70 between its ATP and ADP conformations during assembly of glucocorticoid receptor.hsp90 heterocomplexes.</a> Biochemistry. 40, 1109-1116 (2001).</li><li>Murphy, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pifithrin-alpha+inhibits+p53+signaling+after+interaction+of+the+tumor+suppressor+protein+with+hsp90+and+its+nuclear+translocation.">Pifithrin-alpha inhibits p53 signaling after interaction of the tumor suppressor protein with hsp90 and its nuclear translocation.</a> J. Biol. Chem. 279, 30195-30201 (2004).</li><li>Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+dynamics+of+hsp90+action+on+the+glucocorticoid+receptor+by+acetylation/deacetylation+of+the+chaperone.">Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone.</a> J. Biol. Chem. 280, 33792-33799 (2005).</li><li>Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+assembly+of+a+glucocorticoid+receptor&#149;hsp90+heterocomplex+resolves+two+sequential+ATP-dependent+events+involving+first+hsp70+and+then+hsp90+in+opening+of+the+steroid+binding+pocket.">Stepwise assembly of a glucocorticoid receptor&#149;hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket.</a> J. Biol. Chem. 275, 18054-18060 (2000).</li><li>Pratt, W. B., Toft, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+receptor+interactions+with+heat+shock+protein+and+immunophilin+chaperones.">Steroid receptor interactions with heat shock protein and immunophilin chaperones.</a> Endocr. Rev. 18, 306-360 (1997).</li><li>Bodwell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+phosphorylated+sites+in+the+mouse+glucocorticoid+receptor.">Identification of phosphorylated sites in the mouse glucocorticoid receptor.</a> J. Biol. Chem. 266, 7549-7555 (1991).</li><li>Penna, A., Cahalan, M. W. e. s. t. e. r. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blotting+using+the+Invitrogen+NuPage+Novex+Bis+Tris+minigels.">Blotting using the Invitrogen NuPage Novex Bis Tris minigels.</a> J. Vis. Exp. (7), e264-e264 (2007).</li><li>Choo, Y. S., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Protein+Ubiquitination.">Detection of Protein Ubiquitination.</a> J. Vis. Exp. (30), e1293-e1293 (2009).</li><li>Murphy, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+mechanism+of+assembly+of+a+glucocorticoid+receptor&amp;bull;Hsp70+complex+that+is+primed+for+subsequent+Hsp90-dependent+opening+of+the+steroid+binding+cleft.">Visualization and mechanism of assembly of a glucocorticoid receptor&amp;bull;Hsp70 complex that is primed for subsequent Hsp90-dependent opening of the steroid binding cleft.</a> J. Biol. Chem. 278, 34764-34773 (2003).</li><li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. 389, pl2-pl2 (2007).</li><li>Davis, T. R., Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IP-FCM:+Immunoprecipitation+Detected+by+Flow+Cytometry.">IP-FCM: Immunoprecipitation Detected by Flow Cytometry.</a> J. Vis. Exp. (46), e2066-e2066 (2010).</li><li>Felts, S. J., Karnitz, L. M., Toft, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functioning+of+the+Hsp90+machine+in+chaperoning+checkpoint+kinase+I+(Chk1)+and+the+progesterone+receptor+(PR).">Functioning of the Hsp90 machine in chaperoning checkpoint kinase I (Chk1) and the progesterone receptor (PR).</a> Cell Stress Chaperones. 12, 353-363 (2007).</li><li>Cintron, N. S., Toft, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+requirements+for+Hsp40+and+Hsp70+in+the+Hsp90+chaperone+pathway.">Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway.</a> J. Biol. Chem. 281, 26235-26244 (2006).</li></ol>

选择电泳试剂和图像分析软件是由Bio - Rad公司提供的。

上文所述的检测可以适应测试无数影响hsp90/hsp70-based伴​​侣机械伴侣行动以及GR类固醇具有约束力的条件。这是以前报道的方法 4,8,9,17的修改，旨在充分利用电泳技术的最新进展，并接触到更广泛的研究界。可以添加额外的辅助因子或蛋白质的利益，在GR•HSP90 heterocomplex重组的混合物，在本议定书第4步中所述，以观察对类固醇的约束力，heterocomplex蛋白质相互作用，并共同因素的要求影?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> Sf9细胞</td><td> Invitrogen公司</td><td></td><td>杆状病毒表达的重组小鼠GR </td></tr><tr><td> L929细胞</td><td> ATCC </td><td> CRL - 2173 </td><td>内源性鼠标GR细胞质准备（替代杆状病毒表达） </td></tr><tr><td> FiGR杂交瘤细胞细胞</td><td> ATCC </td><td> CRL - 2173 </td><td>准备含有反GR单克隆抗体的腹水（替代纯化BuGR2抗体） </td></tr><tr><td>完整的迷你蛋白酶抑制剂，EDTA无</td><td>罗氏应用科学部</td><td> 11836170001 </td><td></td></tr><tr><td>蛋白A琼脂糖</td><td> Sigma - Aldrich公司</td><td> P3391 </td><td></td></tr><tr><td>兔网织红细胞裂解液</td><td>绿色公顷（俄勒冈州，威斯康星州） </td><td></td><td>内源性hsp90/hsp70伴侣机械的丰富来源</td></tr><tr><td>肌酸磷酸激酶</td><td> Sigma - Aldrich公司</td><td> C3755 </td><td> ATP再生系统</td></tr><tr><td>磷酸肌酸</td><td> Sigma - Aldrich公司</td><td> P7936 </td><td> ATP再生系统</td></tr><tr><td>反GR鼠单克隆抗体（BuGR2） </td><td>皮尔斯/ Thermo Scientific的</td><td> MA1 - 510 </td><td>用于遗传免疫吸附作为第一抗体免疫印迹</td></tr><tr><td>抗- HSP90鼠单克隆抗体（AC88） </td><td>恩佐生命科学</td><td> ADI公司的SPA - 830 </td><td>作为主要抗体用于免疫印迹</td></tr><tr><td>抗HSP70鼠单克隆抗体（N27F3 4） </td><td>恩佐生命科学</td><td> ADI公司的SPA - 820 </td><td>作为主要抗体用于免疫印迹</td></tr><tr><td>从小鼠血清IgG抗体</td><td> Sigma - Aldrich公司</td><td> 038K7690 </td><td>作为非免疫抗体用于免疫吸附阴性对照</td></tr><tr><td>抗鼠IgG -过氧化物酶的共轭</td><td> Sigma - Aldrich公司</td><td> A4416 </td><td>作为第二抗体用于免疫印迹</td></tr><tr><td>的Experion微流体电泳系统</td><td> Bio - Rad公司</td><td> 7007001 </td><td>替代传统的SDS - PAGE </td></tr><tr><td> [1,2,4,6,7 - 3H]地塞米松</td><td>珀金埃尔默</td><td> NET1192001MC </td><td>按照安全防范措施</td></tr></tbody></table>

biochemical reconstitution of steroid receptor&#x2022;hsp90 protein  complexes and reactivation of ligand binding

HSP90是一个重要和非常丰富，已发现调节超过150个的真核细胞信号蛋白，包括转录因子（如核受体，P53），并参与细胞周期蛋白激酶（如SRC，英国皇家空军，Akt激酶）的分子伴侣蛋白，肿瘤的发生，细胞凋亡和多种真核细胞信号转导通路1,2。许多这些“客户”HSP90蛋白，类固醇受体大会•HSP90复合物是最好的定义（ 图1）。我们在座的适应性强的糖皮质激素受体 ​​（GR）的免疫测定和体外GR•HSP90重组的方法，可随时用来探测HSP90真核细胞的功能活动，HSP90介导的类固醇激素受体 ​​配体的结合，以及分子伴侣辅助因子的要求。例如，此法可用于测试HSP90的辅助因子的要求和加入外源性化合物的重组进程的影响。 遗传资源已经为研究HSP90一个特别有用的系统，必须绑定到HSP90，因为受体有一个开放的配体结合裂访问类固醇 3 。内源性的，unliganded的GR非共价的约束，以HSP90的哺乳动物细胞的细胞质中。如发现内源性GR•HSP90 heterocomplex，GR配体结合裂，是开放的和有约束力的类固醇的。如果从GR HSP90不赞成或如果其功能受到抑制，受体是无法绑定类固醇和需要重组的GR•HSP90 heterocomplex的结合活性类固醇之前恢复 4 。遗传资源，可以从细胞细胞质使用单克隆抗体免疫沉淀，如HSP90蛋白复合物，以遗传资源，可以通过免疫印迹检测。免疫沉淀的GR结合活性的类固醇，可以由孵化与[3H]类固醇immunopellet 。 以前的实验已经证明HSP90介导的GR配体裂约束力的开放需要HSP70，第二个分子伴侣，也为真核细胞的活力至关重要。 HSP90和HSP70的生化活性催化合作伴侣蛋白合，hsp40，和P23 5 。一个多蛋白的伴侣机械HSP90，HSP70，合，和hsp40是在真核细胞的细胞质内源性目前，网织红细胞裂解液提供了一个伴侣，含有丰富的蛋白质来源6。 提出的方法，GR是immunoadsorbed从细胞细胞质和内源性hsp90/hsp70伴侣用温和的盐条件下的机械剥离。盐剥离的GR然后孵育•HSP90 heterocomplex和类固醇的结合活性的激活7，网织红细胞裂解液，三磷酸腺苷，和K +，这在重组的GR结果。可以利用这种方法来测试各种伴侣的辅助因子，新的蛋白质，和实验HSP90或GR抑制剂的影响，以确定其HSP90介导的类固醇具有约束力8-11的功能意义。

Watch this Scientific Journal Video about Biochemical Reconstitution of Steroid Receptorâ€¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding at JoVE.com

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

一个<em&gt;在体外</em&gt;准备从纯化的蛋白质和细胞裂解液的功能性糖皮质激素受体（GR）•HSP90蛋白复合物的方法描述。该方法利用免疫吸附的盐剥离和蛋白质复合体重组重组的GR。辅助因子和缓冲条件的重要性进行了讨论，作为潜在的方法应用。

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

biochemical-reconstitution-steroid-receptorhsp90-protein-complexes

Research

JoVE Journal

Biology

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

16.3K 次观看.  Seattle University. 一个在体外准备从纯化的蛋白质和细胞裂解液的功能性糖皮质激素受体（GR）•HSP90蛋白复合物的方法描述。该方法利用免疫吸附的盐剥离和蛋白质复合体重组重组的GR。辅助因子和缓冲条件的重要性进行了讨论，作为潜在的方法应用。

视频: Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

科研

生物学

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

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

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

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

蛋白质纯化方法测定结合亲和力微尺度热泳

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

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

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 ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.