This work was supported by the European research council by a ERC starting/consolidator grant to RC and by the Dutch Foundation for research on matter (FOM) via a FOM program (10TM01).

1，总色素的提取菠菜叶<ol><li>均质化1一把菠菜叶（约20克）在100毫升冷水研磨缓冲液的使用混合器，持续20秒（ 见表1）。 </li><li>筛选通过尼龙布的两层的溶液用20微米和离心机滤液中的气孔直径为1,500 xg离心4℃10分钟。 </li><li>重悬含有用软艺术家叶绿体沉淀涂料刷在1ml冷洗涤缓冲液（ 见表1）。一旦沉淀物再悬浮，在4℃下加入50mL洗涤缓冲液和离心的溶液以10,000×g离心10分钟。 </li><li>除去上清液，并轻轻悬浮在50ml洗涤缓冲液的粒料（囊体）（ 见表1）。 </li><li>离心机在4℃下将溶液以10,000 xg离心10分钟，完全除去上清液。在这一点上，进行在黑暗中执行以下步骤，以避免颜料氧化。 </LI&gt; <li>加入〜20毫升80％丙酮缓冲用Na 2 CO 3（ 见表1）以提取颜料。留在冰液10分钟，偶尔涡旋。 </li><li>沉淀的细胞成分通过离心，12,000 xg离心15分钟，在4℃下。 注意：如果颜料不完全提取，沉淀都会有绿色和步骤1.6应重复。 </li><li>收集上清液到分液漏斗中。添加0.4体积乙醚，剧烈震荡并打开阀门泄了气。 </li><li>加入0.8倍体积的0.33 M氯化钠和剧烈混合。允许〜10分钟的层分离。在上面的醚相包含所提取的颜料。取下透明的较低阶段。 请注意：如果分离是不明确的，冻结和解冻的溶液，以改善相分离。 </li><li>从分液漏斗顶部倒出到一个合适的玻璃容器中移除醚。干，加入大一勺ular无水硫酸钠。摇动溶液，并允许〜5分钟，使干燥剂，从乙醚中吸收水分。 注意：重​​复此步骤，如果硫酸钠的出现彻底成群在一起;应该有一定的自由浮动的结晶，当醚充分干燥。如果水层的形式，增加额外的无水硫酸钠前，用巴斯德吸管删除此。 </li><li>倒出醚，以一个新的玻璃容器中，在离开硫酸钠固体的后面。 </li><li>蒸发乙醚在旋转SpeedVac中或在N个2流。 </li><li>在10毫升100％丙酮中完全溶解色素。 </li><li>稀释少量（〜3微升）在1ml 80％的丙酮中，并测量吸收光谱并确定叶绿素a / b比值，并与由Porra 等人所述的方法中的叶绿素浓度。 （1989）15。 </li><li>等分试样并干燥在旋转SpeedVac中或在N 2流中的颜料，直至丙酮是完全蒸发。储存干颜料在-80℃。 </li></ol> 2，提取类胡萝卜素的菠菜<ol><li>按照步骤1.1〜1.5。在这一点上，进行在黑暗中执行以下步骤，以避免颜料氧化。 </li><li>重悬在〜50毫升96％乙醇的缓冲用Na 2 CO 3（ 见表1），提取该色素的类囊体颗粒。留在冰上的溶液中5分钟。 </li><li>沉淀的细胞成分通过离心，12,000 xg离心15分钟，在4℃下。 注意：如果颜料不完全提取，沉淀都会有绿色和步骤2.2应重复。 </li><li>收集上清液，加入0.1体积的80％的KOH（重量/体积），以引发皂化反应。 </li><li>离开该溶液在4℃的CO / N，盖紧，并从光保护。 </li><li>收集的溶液放入分液漏斗中。加入1倍体积的乙醚，轻轻混匀。 </li><li>添加0.8体积0.33 M氯化钠超微电极，轻轻混匀。允许〜10分钟的层分离。在上面的橙色醚相含有皂化的类胡萝卜素。通过漏斗的旋塞阀排水除去绿色下层相。 </li><li>加入3倍体积的水，轻轻混匀，以除去氢氧化钾。让各层分开。注意：如果上期出现浑浊，氯化钠加入少量（ 如 3克氯化钠200毫升的溶液），轻轻摇晃溶解。 </li><li>通过漏斗的旋塞阀排水除去下层相。 </li><li>按照步骤1.10至1.13。 </li><li>稀释少量（〜3微升）在1ml 80％的丙酮中，并测量吸收光谱在80％丙酮440纳米。以确定的浓度，使用平均系数灭绝的类胡萝卜素（ε440 = 255）16，在下面的公式：车载[毫克/毫升] =（绝对440纳米 / 225）×11（光路）= 1厘米。 </li><li>分装和干燥carotenoiDS在SpeedVac中或在氮气流中，直到所有的乙醚被蒸发。储存干颜料在-80℃。 </li></ol> 3，总色素和类胡萝卜素的提取莱茵衣藻 <ol><li> C.成长通过散布少量液体培养在表面上在培养皿中藻在固体培养基咨询17。 20微摩尔的照片PSA米-2秒的连续光照通量增长-1直到细胞的绿色层是可见的。 </li><li>用无菌接种环，收获了少量C的藻从固体TAP培养基中，并把该单元到500ml丝锥介质17中的1L烧瓶中。成长的文化，在25℃，170转搅拌20微摩尔的照片PSA米-2秒的连续光照通量-1。 </li><li>后5-6天，培养物应达到对数生长期结束时（6×10 6个细胞/ ml的或在750纳米2-2.5光密度）。离心文化为4,000×g离心15分钟，在4℃。 </li><li>总色素的提取，按照步骤1.6至1.15。 </li><li>总色素提取物500毫升C的充分发展文化，产莱茵衣藻是大约5毫升0.5毫克叶绿素A + B /毫升浓度的溶液。 </li><li>类胡萝卜素的提取，按照步骤2.2至2.12。 </li></ol> 4，净化包涵体<ol><li>克隆感兴趣的LHC蛋白的编码序列插入一个表达载体中使用标准分子生物学程序，结果在一个稠合的C-端His标签。把这种构建到大肠杆菌大肠杆菌宿主菌株如BL21（DE3）。 </li><li>制备裂解缓冲液，洗涤剂缓冲器，加入Triton缓冲液，TE（ 表1），1M异丙基β-D-1-硫代半乳糖苷（IPTG）和LB与适当的抗生素的培养基18。 </li><li>挑上为单ê 大肠杆菌大肠杆菌菌落含有新鲜的划线盘的表达克隆入〜5ml的LB培养基中使用的标准方法6的合适的抗生素。生长于37℃下用220rpm的转速搅拌至少16小时。 </li><li>添加2.5毫升的O / N培养成1升锥形瓶中，用250毫升的LB辅以适当的抗生素。 </li><li>生长细胞为2-3小时（或直到OD 600为〜0.6），在37℃下以220rpm。 </li><li>添加IPTG至1毫米的终浓度。继续以220rpm的转速3-4小时生长的细胞在37℃。 </li><li>离心培养10分钟，以5,000 XG在4℃下在预先称重的离心管中。彻底弃去上清液，并确定重粒料通过再次称重并减去该离心管重量。 </li><li>悬浮大肠杆菌大肠杆菌细胞沉淀于0.8ml / g的裂解缓冲液，剧烈涡旋。 注意：可选地，将细胞沉淀可以在-80冻结下以后使用。如果启动了冻颗粒，允许加入裂解液之前完全解冻。 </li><li>加入2毫克，每克细胞的溶菌酶，并孵育在湿冰偶尔涡旋30分钟。 </li><li>加入20微克/毫升DNA酶，10毫米氯化镁2，1 mM氯化钠，20微克/毫升RNA酶。涡旋并置于冰上30分钟。 </li><li>加入2mL每克细胞的冷洗涤剂缓冲。搅拌均匀，保持室温5分钟。 </li><li>转移至2ml离心管（分成两管，如果需要的话）。离心机以12,000 XG 10分钟，在4℃下沉淀的包含体。 </li><li>加入1毫升冷的Triton缓冲液和悬浮完全由声波处理的颗粒（3脉冲X 5秒×50％的电力与20秒的间隔）。注：具有管在小烧杯用冰水环绕声处理过程中保持低温。在多重管的情况下，结合的悬浮包涵体成一个管重悬浮后。 </li><li>离心机以12,000 10分XG在4℃下沉淀的包含体。 </li><li>重复步骤4.13和4.14两次。 </li><li>悬浮在1毫升冷的TE与声波处理包涵体作最后的洗涤，除去海卫缓冲区。离心机以12,000 XG 10分钟，在4℃下沉淀的包含体。 </li><li>悬浮颗粒在1毫升冷的TE通过声波处理。 </li><li>评估蛋白质浓度通过标准方法如Bradford测定19。在-20℃的包涵体店等分。 </li></ol> 5，重组这个协议典型地产生1-2毫升重组蛋白以4的OD时测定吸光度在的Qy区域（600-750纳米）。量可以根据需要调节，虽然应注意，以在手术过程中保持适当的比率。 <ol><li>制备如表1中所述的下列方案：2X重建缓冲液，20％的OG，2米氯化钾，TE中。执行后续的sTEPS在昏暗的光线。 </li><li>悬浮800微克的LHC的包涵体中共有400μL的TE在2 ml离心管中。加入400μL的2倍重组缓冲区和涡短暂的。 </li><li>添加0.6μLβ-巯基乙醇（股票14.8米）有10毫米的终浓度。加热该蛋白为在98℃下1分钟。短暂涡旋并置于室温3分钟。 </li><li>重悬在30微升100％的乙醇500微克总干叶绿素色素加80微克类胡萝卜​​素由剧烈振荡1分钟，或发生在洗澡超声仪1-2分钟。 </li><li>旋转的颜料组合〜30秒在15,800 XG在4℃下，并确认没有沉淀。如果有一个小球，重复涡旋和/或超声处理。重要：重悬浮和旋转之后，马上添加颜料的蛋白质，或者它可以聚合和需要被再次悬浮。 </li><li>慢慢地将颜料混合添加到冷却的蛋白质而涡旋。继续涡5到10秒钟EC和地方管湿冰上。要小心，不要涡旋太大力的蛋白质可以溢出管的顶部。 </li><li>加入94微升20％辛基β-D-吡喃葡糖苷（OG）（终浓度2％），短暂涡旋并保持在冰上10分钟。 </li><li>加入90微升的氯化钾2米（终浓度为150〜200毫米），轻轻振荡并保持在冰上20分钟。注：列准备（第6节）可以在这个时候启动。 </li><li>自旋为在15,800 XG 10分钟，在4℃。除去上清液，而不会干扰沉淀（沉淀LDS）到10毫升管。保持冷并避光。 </li></ol> 6，镍柱纯化<ol><li> OG缓冲器，OG漂洗缓冲液，洗脱缓冲液：如表1中所述制备以下的溶液。 </li><li>镍 - 琼脂糖凝胶柱（1毫升）或等效连接到蠕动泵以确保没有空气进入柱内时本步骤和下面的步骤。 </li><li>集合T的速度他泵送至1毫升/分钟，冲洗柱，用5-10毫升的水以除去存储解决方案。 </li><li>平衡用3-4毫升的OG缓冲柱。 </li><li>加入3-4毫升的OG缓冲器的蛋白质样品并加载到柱上。注意：如果蛋白质已除去LDS后一直坐在冰上超过10分钟，再次旋转在15,800 XG在4℃下1分钟，以去除任何额外的LDS沉淀。 </li><li>冲洗用5ml OG缓冲柱。 </li><li>冲洗该柱用2毫升的OG漂洗缓冲液。 </li><li>洗脱用3毫升的洗脱缓冲液将结合的蛋白质。收集绿色洗脱包含重新构造蛋白质。注意：这是一般在总约1ml。 </li></ol> 7，蔗糖密度梯度离心<ol><li>蔗糖溶液，0.06％β-DM，0.01M HEPES，pH值7.6：如表1中所述制备以下的溶液。 </li><li>填写超速离心机管道，并在-20°股份有限公司/ N澳蔗糖溶液和冻结ř-80℃下放置至少1小时。 </li><li>从冰箱中取出试管并允许解冻原状，在4℃。注：冷冻/解冻过程产生的梯度从0.1至1M蔗糖。 15毫升管通常解冻约3小时。 </li><li>从顶部小心的取出相同体积的镍琼脂糖凝胶柱在步骤6.8洗脱的绿色部分。然后加载在上面的重组样品缓慢以避免扰乱梯度。 </li><li>使用SW-41或SW-60水平转头18小时，设定为慢加速和停止没有刹车平衡管和自旋以200,000 XG在4℃下在超速离心机。 </li><li>小心地取出从用钳子管托的梯度。用注射器用长针，有一个钝开来收集从顶部的分数。注意：可替换地，通过刺穿管子用针和收集滴收集从底部馏分。 </li></ol>

The authors declare no conflict of interest

膜蛋白是不那么容易学习。天然膜蛋白的分离中所需要增溶脂质双层带洗涤剂，它可以破坏蛋白质和除去必要的辅因子复杂。这些蛋白质也可能含有低含量的生物膜，或与密切相关的蛋白质混合时，如在光捕获复合物的情况下，这使得单个复合物难以纯化。在大肠杆菌中的异源蛋白的表达大肠杆菌和体外重组 提供了避免这些问题的可能性。 在体外重组和折叠的蛋白纯化的结果在于，具备特征非常类似于天然复合物20,21,23，因而可用于研究，不能纯化至均质24复合物- 27。 此方法使用菠菜，这是很容易attainab乐全年，作为总色素和类胡萝卜素制剂的来源。蛋白质天然藻类的一些reconstitutions，使用颜料从藻类提纯的优选由于不同颜料的组合物。不管色素源的叶绿素 a / b比值，叶绿素/车比例保持不变。 要认识到，重组的效率通常为约35％，28是很重要的。因此，有必要从重构后的溶液中除去未结合的色素和展开的脱辅基蛋白。两步纯化方案，提出在此协议（也见结果）。然而，应该指出的是，蔗糖梯度步骤不允许载脂蛋白和全息蛋白的完全分离。对于大多数分析，这是没有问题的，因为脱辅基蛋白不含有颜料，因而不与功能性的测量产生干扰。然而，如果有必要从FR完全去除脱辅基蛋白含有该重组复合物（例如，为了计算颜料与蛋白质的化学计量）的动作，阴离子交换柱可以使用（参见Passarini 等 ，2009 29详细说明）。 的能力，以重折叠的重组捕光蛋白与分离的颜料在体外提供一个机会通过修改重建“环境”以不同的方式，从而改变所得到的复合体的特征为“操纵”的复合物。例如，重组过程中改变了色素成分可能会导致与改变色素成分复杂。此功能可以用于研究影响各种颜料对配合物的结构和稳定性。通常是从菠菜中获得的颜料制剂有3 叶绿素 a / b比值：1和2.9 CHL /汽车之比为2:1。该比率通常会产生改组后的蛋白质具有相同属性的第native之一。然而，调整的叶绿素a / b比值，加入纯化的叶绿素a或 b的可能影响因不同的结合位点30的选择性不同颜料的结合- 33。这是可能的，因为大多数的颜料结合位点也不是完全选择性的叶绿素a或叶绿素 b，而是可以容纳两个，虽然以不同的亲和力10,30,34。以类似的方式，将类胡萝卜素的结合位点也被示出，以便能够容纳一个以上的叶黄素物种8,35 - 38。 CP26，高等植物，使用各种颜料的组合物的另一种颜料-蛋白质复合物的不同reconstitutions示于表2 39。这些reconstitutions被用来评估的结合位点的特定颜料39的亲和力。有趣的是要注意，为了得到具有相同的色素c进行复杂omposition为原生的，颜料混合的叶绿素 a / b值必须是3:1。这似乎是高等植物20,40的所有LHC配合物的情况下。 分子生物学的重组技术的组合允许在更详细地研究了叶绿素结合复合物的性质。 44 -上的配合物，或它们中的蛋白-蛋白相互作用参与的稳定性和折叠的不同蛋白质结构域的重要性，已通过截断的脱辅基蛋白或进行随机诱变8,41决定。 52 -单个氨基酸残基的不同颜料的协调重要可以通过位点定向诱变，以分析个别颜料的性质或评估其对复杂10,28,29,45的功能和稳定性的贡献被改变。 图6示出重构Lhcb4（CP29）与组氨酸的突变位216 53野生型和突变体复合物的颜料组合物的比较表明，该突变导致1叶绿素损失的分子，表明该目标部位容纳有叶绿素a在WT复杂。 WT和突变体的吸收光谱的差异，根据归一化的颜料含量，还示出了丢失的色素的吸收特性。在这种情况下，差可以看出，在680nm处的主峰，表明叶绿素a通过His216协调吸收这个波长（关于该突变体更多细节和光谱性质看莫佐等 ，2008 53）。突变分析也可用于确定环境对颜料54的光谱特性的影响。 总之，捕光蛋白可容易地在体外重建导致颜料的proteiÑ​​配合非常相似的性质天然复合物。以这种方式，分离的天然蛋白的困难被消除，同时还提供蛋白质制剂具有高产率和高纯度的进一步研究。一3的重要性：1 叶绿素 a / b比值在制造复杂的地道强调，并提供了被重组野生型和突变型LHCs的例子来说明该技术的应用。

植物和藻类的光合作用装置，包括结合叶绿素 a（ 叶绿素 a）整合膜蛋白，B（叶绿素b）和类胡萝卜素（CAR）。这些色素蛋白复合物活性在收获光能和传送该激发能量向反应中心，在那里它被用于促进电荷分离2。他们还参与了保护高光损伤3,4光合机构的监管反馈机制。捕光复合物（LHCs）是由植物和藻类5大家族相关的蛋白质的。 该家族的每个成员的均相提纯变得复杂化的复合物的高度相似的化学和物理性质。此外，纯化方法通常会导致颜料或其它潜在的辅因子如脂类的损失， 在体外重组代表中TS的有力的方法来克服这些问题。与光系统II（LHC-II）相关联的LHC首先在体外通过Plumley和施密特重构于1987 1。研究者分别萃取脱脂蛋白和颜料从植物叶绿体，然后结合颜料的热变性蛋白在锂的存在下十二烷基硫酸盐（LDS），其次是冷冻和解冻1的三个周期。它们表明，重新构成的LHC配合物的光谱特性非常相似，配合物从植物纯化。再造LHC色素蛋白复合物，可能是由于一些固有的自组装特性，以及从生物中分离纯化物的难度容易，导致了快速采用其他研究人员的方法。过量表达大肠杆菌 （ 大肠杆菌 ），光合蛋白的重建是由保尔森和他的同事在1990年6实现的。在大肠杆菌大肠杆菌，过度膜蛋白通常包含在包涵体，其设施的净化。重构是通过含有重组蛋白在LDS的存在下，包涵体，然后加入颜料，它启动了蛋白质折叠的热变性来实现。折叠LHCII复杂的是一个两步骤的过程：首先， 叶绿素a，势必在少于1分钟;第二， 叶绿素 b的约束，并稳定在几分钟内7。 除了 ​​提供洞察折叠动力学， 在体外重组结合位点定向诱变已允许的特定氨基酸对稳定性很重要（ 例如，8,9）或颜料协调的标识（ 例如，10）。复性，通过调整参数，如颜料的组合物或清洁剂的条件的操作还确定元素critica升的适当折叠，如叶黄素为LHCII复杂的要求（ 例如，1,11）。此外，结合到复合物个体的颜料的特性调查使用复合物重新溶解在体内有可能（ 例如，10）。 这里所描述的方法开始的颜料从菠菜隔离（叶绿素和类胡萝卜素）和绿藻莱茵衣藻。从E来 LHC蛋白的表达和纯化大肠杆菌中包涵体的形式，然后详细描述的，其次是LHC的重构和随后的纯化用Ni亲和柱。在最后的步骤中，将重建的复合物是通过蔗糖梯度离心进一步纯化以除去游离的颜料和非折叠脱辅基蛋白。该协议代表了一个优化过程结合了若干修改已推出了不同的实验室对时间1,6,10,12 -14。

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;HisTrap HP</td>
			<td style="width:115px;">GE Healthcare</td>
			<td>17-5247-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon cloth&#160;</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td>20 &#956;m pores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soft artists paint brush&#160;</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NONIDET P-40</td>
			<td style="width:115px;">Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beta-DM</td>
			<td style="width:115px;">Sigma</td>
			<td>D4641</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DNAase</td>
			<td style="width:115px;">ThermoScientific</td>
			<td>EN0525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Milk Powders</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNAase</td>
			<td style="width:115px;">ThermoScientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Octyl &#946;-D-glucopyranoside</td>
			<td style="width:115px;">Sigma</td>
			<td>O8001&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultracentrifuge XL</td>
			<td style="width:115px;">Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TAP medium</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td>see reference 17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB medium</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td>see reference 19</td>
		</tr>
	</tbody>
</table>

in vitro reconstitution of light-harvesting complexes of plants and green algae

在植物和绿藻，光被光捕获复合物（LHCs），一个家庭的整合膜蛋白的坐标叶绿素和类胡萝卜素的捕获。 在体内 ，这些蛋白质折叠和颜料以形成插入到类囊体膜复合物叶绿体。高的相似性，在家庭成员的化学和物理性质，与事实，他们可以隔离期间容易失去色素在一起，使他们在原生状态的净化有挑战性。一种替代的方法来获得LHCs的均质制剂是由Plumley和施密特于1987年1发达，谁表明，它能够重构这些配合物在体外从纯化的颜料和非折叠脱辅基蛋白启动，从而导致配合性质非常类似于天然复合物。这开辟了道路使用细菌表达的重组蛋白在体外 </eM&gt;重建。该重建方法是有力各种原因：（1）可以得到各个复合物的纯制剂，（2）颜料组合物可被控制，以评估其贡献的结构​​和功能，（3）的重组蛋白可以被突变，以研究该官能个别残基（ 例如，颜料的结合位点）或蛋白质结构域（ 例如，蛋白质-蛋白质相互作用，折叠）的作用。此方法已被优化在几个实验室，并应用于大多数的光捕获复合物。这里所描述的协议细节重构目前在我们的实验室中使用的体外光捕获复合物的方法， 并提供描述该方法的应用例子。

该协议的细节的方法来重建叶绿素-a a / b结合蛋白在体外 。此技术允许这些色素蛋白复合体外从脱辅基蛋白，其可以通过过表达的异源系统中得到起始，并从植物或藻类中提取色素的折叠。重构后，复性色素 - 蛋白质复合物是由过量的颜料和在两个步骤中展开的脱辅基蛋白纯化。在第一步骤（ 图1 AB）是基于His标签的蛋白质，其允许在除去未结合的色素的很大一部分的C末端的存在。第二纯化步骤采用蔗糖密度梯度离心，（ 图2），其中未折叠蛋白通常迁移比含重组蛋白的绿色带慢。 在体外重建的目标是获得复合物具有相同的正确领带作为原生的。为了说明这个结果，对在体内光捕获复合物的光谱特性与所述相同的LHC复杂的重构的体外 13,20,21相比较。该LHCs的在可见光范围内（350纳米和750纳米）的吸收光谱依赖于复杂的颜料组合物，以及所述颜料的环境（包括蛋白质），它是这样一个敏感的工具来检查质量的重建。在图3中 ，CP24的吸收光谱中，叶绿素a / b结合来自拟南芥 ，重构的体外蛋白，是具有相同的复杂的纯化的拟南芥类囊体21的光谱进行比较。在光谱，所以能够识别的Qy和叶绿素的 （峰在439分之671纳米）的索瑞过渡和叶绿素 b（在466分之649处的峰）。本机与重组复合显示了相同ABSOrption光谱，显示出几乎相同的颜料成分和组织。荧光光谱法可用于评估重构复合物的质量。荧光发射光谱中不同波长的，它激发不同的优先色素测激励时： 叶绿素 a在440nm，叶绿素b在475纳米，和叶黄素在500纳米。在正确折叠的蛋白质的颜料复合物，叶绿素b和叶黄素在几皮 ​​秒其激发能转移主要叶绿素a，并从产生的单峰具有相同的形状和最大值在全部三个激发的热平衡系统的荧光起源波长（ 图4A-B）。叶绿素乙未配位到蛋白质的存在可以通过一个额外的峰或肩围绕在475 nm激发（ 图4C）为650nm的认可。无叶绿素的存在，而不是导致到周围675纳米，这是在440纳米激发主要存在额外发射。在这两种重组的475 nm激发和天然CP24配合物（ 图4D）的荧光发射光谱显示出一个单峰在681纳米，表示重新构成的复合物被正确折叠。该色素蛋白复合物被正确地重新组织的附加确认来自圆二色性（CD）测量。在可见光区域的CD信号取决于颜料之间的激子的相互作用，它是这样来的发色团22的组织的微小变化非常敏感， 图5示出的重组和天然CP24的CD光谱，与在典型的指纹峰681纳米，650纳米和481纳米。总之，本机的光谱性质和重组CP24证实，重组过程收益率类天然复合吹田之间的高度相似性<html>竹叶提取用于捕光蛋白在体外研究中。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51852/51852fig1highres.jpg" width="500" /> 图1表示重组LHC的蛋白质与利用镍柱His标签的净化。（a）在净化，His标签的蛋白，既包括重组复合物（绿色六边形）和未复原/聚集蛋白（橙色六边形）绑定到镍琼脂糖（蓝点）的表面，而绑定颜料（彩色小点）流过。 （B）当该柱用含咪唑的洗脱缓冲液，再溶解和未重组蛋白是通过收集在流。 hres.jpg“WIDTH =”500“/&gt; 图2蔗糖重组LHCII的梯度纯化以镍柱后的重构复合物从游离颜料的密度梯度分离。墨绿色条带代表重新构造LHCII和浅绿色的背景是由无色素。 <img alt="图3" fo:content-width="4in" src="/files/ftp_upload/51852/51852fig3highres.jpg" width="400" /> 图3吸收重组蛋白CP24（rCP24，红色线）和天然酮（nCP24，黑线）从拟南芥中分离的光谱。在这两个光谱，所以能够识别的Qy和叶绿素a的索瑞过渡（峰在439分之671纳米）和叶绿素 b（在四百六十六分之六百四十九波长峰值）。这个数字已经被修改Passarini 等 。 2014年21。 图4的荧光发射光谱。的重组CP24野生型复合物（A）和归一化到最大（B）中示出从叶绿素b和Xanthophyls有效的能量转移到叶绿素a的荧光发射光谱。重组CP24（rCP24）和乡土情结（nCP24）从拟南芥中分离的（C）的荧光发射光谱，光谱归一化的峰值（D）的最大值。 <a href="https://www.jove.com/files/ftp_upload/51852/51852fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51852/51852fig5highres.jpg" width="500" /> 图5圆二色光谱。复溶CP24（rCP24，红色线）和天然复合物（nCP24，黑线）从拟南芥中分离示出非常相似的光谱。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51852/51852fig6highres.jpg" width="500" /> 的CP29野生型（CP29_WT）和突变CP29（CP29_A2）图6的吸收光谱的绿线表示两个图之间的差异。 <table border="0" cellpadding="0" cellspacing="0" tbody="" width="664"><tbody><tr height="22"><td colspan="2" height="22" style="height:22px;width:399px;">所有的缓冲液可以储存于4℃。 </td><td style="width:144px;"></td><td style="width:121px;"></td></tr><tr height="25"><td height="25" style="height:25px;"></td><td> 组件 </td><td> 终浓度 </stron克&gt; </td><td> 其他注意事项 </td></tr><tr height="21"><td height="106" rowspan="5" style="height:106px;"> 研磨缓冲 </td><td>山梨糖醇</td><td> 0.4M的</td><td style="width:121px;"></td></tr><tr height="21"><td height="21" style="height:21px;">甘氨酸</td><td> 0.1M的</td><td style="width:121px;"> pH值7.8 </td></tr><tr height="21"><td height="21" style="height:21px;">氯化钠</td><td> 10毫米</td><td style="width:121px;"></td></tr><tr height="21"><td height="21" style="height:21px;"> 氯化镁 </td><td> 5毫米</td><td style="width:121px;"></td></tr><tr height="22"><td height="22" style="height:22px;">奶粉</td><td> 0.5％w / v的</td><td style="width:121px;"></td></tr><tr height="21"><td height="64" rowspan="3" style="height:64px;"> 洗涤液 </td><td>山梨糖醇</td><td> 50毫米</td><td></td></tr><tr height="21"><td heiGHT =“21”的风格=“高度：21px;”&gt;甲基甘氨酸</td><td> 5毫米</td><td style="width:121px;"> pH值7.8 </td></tr><tr height="22"><td height="22" style="height:22px;"> EDTA </td><td> 10毫米</td><td style="width:121px;"> pH值为8 </td></tr><tr height="21"><td height="64" rowspan="3" style="height:64px;"> 裂解液 </td><td>三</td><td> 50毫米</td><td style="width:121px;"> pH值为8 </td></tr><tr height="21"><td height="21" style="height:21px;">蔗糖</td><td> 2.5％w / v的</td><td></td></tr><tr height="22"><td height="22" style="height:22px;"> EDTA </td><td> 1毫米</td><td style="width:121px;"> pH值为8 </td></tr><tr height="21"><td height="127" rowspan="6" style="height:127px;"> 洗涤缓冲液 </td><td>氯化钠</td><td> 200 mM氯化钠</td><td></td></tr><tr height="21"><td height="21" style="height:21px;">脱氧胆酸</td><td> 1％w / v的</td><td></td></tr><tr height="21"><td heighT =“21”的风格=“高度：21px;”&gt; NONIDET P-40 </td><td> 1％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;">三</td><td> 20毫米</td><td> pH值7.5 </td></tr><tr height="21"><td height="21" style="height:21px;"> EDTA </td><td> 2毫米</td><td> pH值为8 </td></tr><tr height="22"><td height="22" style="height:22px;">的β-巯基乙醇</td><td> 10毫米</td><td></td></tr><tr height="21"><td height="64" rowspan="3" style="height:64px;"> 海卫缓冲 </td><td>的Triton X-100 </td><td> 0.5％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;">三</td><td> 20毫米</td><td> pH值7.5 </td></tr><tr height="22"><td height="22" style="height:22px;">的β-巯基乙醇</td><td> 1毫米</td><td></td></tr><tr height="21"><td height="43" rowspan="2" style="height:43px;"> TE缓冲液 </td><td>三</td><td> 50毫米</td><td> pH值为8 </td><html></tr><tr height="22"><td height="22" style="height:22px;"> EDTA </td><td> 1毫米</td><td> pH值为8 </td></tr><tr height="21"><td height="106" rowspan="5" style="height:106px;"> 重组缓冲区 </td><td> HEPES </td><td> 200毫米</td><td></td></tr><tr height="21"><td height="21" style="height:21px;">蔗糖</td><td> 5％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;"> Lithiumdodecylsulfate（LDS） </td><td> 4％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;">脒</td><td> 2毫米</td><td></td></tr><tr height="22"><td height="22" style="height:22px;">氨基己酸</td><td> 10毫米</td><td></td></tr><tr height="21"><td height="106" rowspan="5" style="height:106px;"> OG缓冲 </td><td>辛基葡糖苷</td><td> 1％w / v的</td><td></td></tr><tr height="21">PX;“&gt;蔗糖</td><td> 12.5％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;">氯化钠</td><td> 0.2M的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;"> HEPES </td><td> 20毫米</td><td></td></tr><tr height="22"><td height="22" style="height:22px;">咪唑</td><td> 10毫米</td><td></td></tr><tr height="21"><td height="64" rowspan="3" style="height:64px;"> OG冲洗缓冲 </td><td>正 - 十二烷基-β-D-麦芽糖苷（β-DM） </td><td> 0.06％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;"> HEPES </td><td> 40毫米</td><td> pH值7.5-9 </td></tr><tr height="22"><td height="22" style="height:22px;">氯化钠</td><td> 0.2M的</td><td></td></tr><tr height="21"><td height="85" rowspan="4" style="height:85px;"> 洗脱缓冲液 </td><td>咪唑</td><td> 0.5M的</td><td></td></tr><tr height=“21”&gt; <td height="21" style="height:21px;">正 - 十二烷基-β-D-麦芽糖苷（β-DM） </td><td> 0.06％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;"> HEPES </td><td> 40毫米</td><td> pH值为8 </td></tr><tr height="22"><td height="22" style="height:22px;">氯化钠</td><td> 0.2M的</td><td></td></tr><tr height="21"><td height="64" rowspan="3" style="height:64px;"> 蔗糖溶液 </td><td>蔗糖</td><td> 20％w / v的</td><td></td></tr><tr height="21"><td height="21" style="height:21px;">正 - 十二烷基-β-D-麦芽糖苷（β-DM） </td><td> 0.06％w / v的</td><td></td></tr><tr height="22"><td height="22" style="height:22px;"> HEPES </td><td> 0.01M的</td><td> pH值7.6 </td></tr><tr height="25"><td height="47" rowspan="2" style="height:47px;width:156px;"> 丙酮80％缓冲与碳酸钠 </td><td>丙酮</td><td> 80％体积/体积</td><td></td></tR&gt; <tr height="22"><td height="22" style="height:22px;">碳酸钠</td><td> 1米</td><td></td></tr><tr height="21"><td height="43" rowspan="2" style="height:43px;width:156px;"> 乙醇96％缓冲与碳酸钠 </td><td>乙醇</td><td> 96％体积/体积</td><td></td></tr><tr height="22"><td height="22" style="height:22px;">碳酸钠</td><td> 1米</td><td></td></tr></tbody></table> 表1列出本协议中使用的缓冲和解决方案。 <table border="0" cellpadding="0" cellspacing="0" width="640"><tbody><tr height="41" style="height:30.75pt"><td class="xl63" height="41" style="height:30.75pt;width:48pt" width="64"></td><td class="xl64" style="width:48pt" width="64"> 叶绿素a 的A / B 混合 </td><td class="xl64" style="width:48pt" width="64"> 叶绿素a <foNT类=“font6”&gt; A / B </td><td class="xl64" style="width:48pt" width="64"> 叶绿素 a </td><td class="xl64" style="width:48pt" width="64"> 叶绿素 b </td><td class="xl64" style="width:48pt" width="64"> 新 </td><td class="xl64" style="width:48pt" width="64"> 紫百合 </td><td class="xl64" style="width:48pt" width="64"> 琵琶 </td><td class="xl64" style="width:48pt" width="64"> CHL TOT </td><td class="xl64" style="width:48pt" width="64"> CHL /汽车 </td></tr><tr height="21" style="height:15.75pt"><td class="xl65" height="21" style="height:15.75pt;width:48pt" width="64"> nCP26 </td><td class="xl66" style="width:48pt" width="64"> - </td><td class="xl66" style="width:48pt" width="64"> 2.2±0.05 </td><td class="xl66" style="width:48pt" width="64"> 6.2<html></td><td class="xl66" style="width:48pt" width="64"> 2.8 </td><td class="xl66" style="width:48pt" width="64"> 0.61 </td><td class="xl66" style="width:48pt" width="64"> 0.38 </td><td class="xl66" style="width:48pt" width="64"> 1.02 </td><td class="xl66" style="width:48pt" width="64"> 9 </td><td class="xl66" style="width:48pt" width="64"> 4.5±0.1 </td></tr><tr height="21" style="height:15.75pt"><td class="xl65" height="21" style="height:15.75pt;width:48pt" width="64"> rCP26 </td><td class="xl66" style="width:48pt" width="64"> 8 </td><td class="xl66" style="width:48pt" width="64"> 2.71±0.05 </td><td class="xl66" style="width:48pt" width="64"> 6.57 </td><td class="xl66" style="width:48pt" width="64"> 2.43 </td><td class="xl66" style="width:48pt" width="64"> 0.72 </td><td class="xl66" style="width:48pt" width="64"> 0.32 </td><td class="xl66" style="width:48pt" width="64"> 0.97 </td>“&gt; 9 </td><td class="xl66" style="width:48pt" width="64"> 3.9±0.04 </td></tr><tr height="21" style="height:15.75pt"><td class="xl65" height="21" style="height:15.75pt;width:48pt" width="64"> rCP26 </td><td class="xl66" style="width:48pt" width="64"> 5.5 </td><td class="xl66" style="width:48pt" width="64"> 2.25±0.05 </td><td class="xl66" style="width:48pt" width="64"> 6.23 </td><td class="xl66" style="width:48pt" width="64"> 2.77 </td><td class="xl66" style="width:48pt" width="64"> 0.77 </td><td class="xl66" style="width:48pt" width="64"> 0.3 </td><td class="xl66" style="width:48pt" width="64"> 0.96 </td><td class="xl66" style="width:48pt" width="64"> 9 </td><td class="xl66" style="width:48pt" width="64"> 4.0±0.1 </td></tr><tr height="21" style="height:15.75pt"><td class="xl65" height="21" style="height:15.75pt;width:48pt" width="64"> rCP26 </td><td class="xl66" style="width:48pt" width="64"> 3 </td><td class="xl66" style="width:48pt" width="64"> 2.08±0.04 </td><td class="xl66" style="width:48pt" width="64"> 6.08 </td><td class="xl66" style="width:48pt" width="64"> 2.92 </td><td class="xl66" style="width:48pt" width="64"> 0.76 </td><td class="xl66" style="width:48pt" width="64"> 0.3 </td><td class="xl66" style="width:48pt" width="64"> 1.04 </td><td class="xl66" style="width:48pt" width="64"> 9 </td><td class="xl66" style="width:48pt" width="64"> 4.1±0.1 </td></tr><tr height="21" style="height:15.75pt"><td class="xl65" height="21" style="height:15.75pt;width:48pt" width="64"> rCP26 </td><td class="xl66" style="width:48pt" width="64"> 1 </td><td class="xl66" style="width:48pt" width="64"> 1.7±0.05 </td><td class="xl66" style="width:48pt" width="64"> 5.7 </td><td class="xl66" style="width:48pt" width="64"> 3.3 </td><td class="xl66" style="width:48pt" width="64"> 0.7 </td><td class="xl66" style="width:48pt" width="64"><html> 0.3 </td><td class="xl66" style="width:48pt" width="64"> 0.9 </td><td class="xl66" style="width:48pt" width="64"> 9 </td><td class="xl66" style="width:48pt" width="64"> 4.3±0.05 </td></tr><tr height="21" style="height:15.75pt"><td class="xl65" height="21" style="height:15.75pt;width:48pt" width="64"> rCP26 </td><td class="xl66" style="width:48pt" width="64"> 0.3 </td><td class="xl66" style="width:48pt" width="64"> 1.11±0.04 </td><td class="xl66" style="width:48pt" width="64"> 4.7 </td><td class="xl66" style="width:48pt" width="64"> 4.28 </td><td class="xl66" style="width:48pt" width="64"> 0.7 </td><td class="xl66" style="width:48pt" width="64"> 0.3 </td><td class="xl66" style="width:48pt" width="64"> 0.9 </td><td class="xl66" style="width:48pt" width="64"> 9 </td><td class="xl66" style="width:48pt" width="64"> 4.2±0.2 </td></tr><tr height="21" style="height:15.75pt">PT;宽度：48pt“WIDTH =”64“&gt; rCP26 </td><td class="xl66" style="width:48pt" width="64"> 0.05 </td><td class="xl66" style="width:48pt" width="64"> 0.23±0.01 </td><td class="xl66" style="width:48pt" width="64"> 1.4 </td><td class="xl66" style="width:48pt" width="64"> 5.6 </td><td class="xl66" style="width:48pt" width="64"> 0.58 </td><td class="xl66" style="width:48pt" width="64"> 0.24 </td><td class="xl66" style="width:48pt" width="64"> 1.11 </td><td class="xl66" style="width:48pt" width="64"> 7 </td><td class="xl66" style="width:48pt" width="64"> 3.1±0.06 </td></tr><tr height="21" style="height:15.75pt"><td class="xl65" height="21" style="height:15.75pt;width:48pt" width="64"> rCP26 </td><td class="xl66" style="width:48pt" width="64"> &lt;0.01 </td><td class="xl66" style="width:48pt" width="64"> 0.11±0.01 </td><td class="xl66" style="width:48pt" width="64"> 0.7 </td><td class="xl66" style="width:48pt" widtH =“64”&gt; 6.3 </td><td class="xl66" style="width:48pt" width="64"> 0.64 </td><td class="xl66" style="width:48pt" width="64"> 0.3 </td><td class="xl66" style="width:48pt" width="64"> 1.08 </td><td class="xl66" style="width:48pt" width="64"> 7 </td><td class="xl66" style="width:48pt" width="64"> 3.06±0.06 </td></tr></tbody></table> CP26祖籍表2颜料含量的复杂相比，重组蛋白色素复合物具有不同的叶绿素A / B比率39。

Watch this Scientific Journal Video about In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae at JoVE.com

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 ...

in-vitro-reconstitution-light-harvesting-complexes-plants-green

Research

JoVE Journal

Biology

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

18.0K 次观看.  VU University Amsterdam. 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.

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

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

科研

生物学

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

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

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

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, ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

依赖的PDZ-CFTR的大分子信号复合物的体外分析

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

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) ...

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

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

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

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

重组β-catenin的降解<em&gt;爪蟾</em&gt;卵提取物

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

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

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 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

单细胞共轭藻类无性系培养的建立

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...