由美国国立卫生研究院授予R01EY018144支持。

1。生产，复性，HPLC纯化全新RBP <ol><li>的pET3a载体，6X His标签上的N-末端的cDNA为人类RBP变换的BL-21cells与。生长转化BL-21细胞在40ml与羧苄青霉素的LB培养基在37℃下摇床直到OD在600nm处达到0.5。诱导RBP蛋白表达加入IPTG至1mm。长出的细菌在37℃下另外5小时。 </li><li> RBP在大肠杆菌中产生的主要是存在于包涵体。为了丰富包涵体在10,000 xg离心20分钟，沉淀细胞。然后超声处理的细胞在5毫升的PBS在冰上与蛋白酶抑制剂，接着通过2个循环的冷冻和解冻。佩莱上下包涵体在20,000 xg离心30分钟，在4℃下去除上清液，并保持在冰上沉淀。 </li><li>通过超声处理在10毫升7.5 M盐酸胍溶解包涵体颗粒。加入一半的体积（5毫升）的25mM的Tris缓冲液，pH值9.0和DTT到最后的C臭氧浓度的的10mM。大力混合溶液在室温下过夜，充分减少和蛋白质变性在旋涡混合器。 </li><li>离心的蛋白质溶液，在4℃下18,000 xg离心20分钟以除去不溶性物质分拆后，将上清转移到新的试管中，并在冰上放置管。 </li><li>准备四个卷（60毫升）的重折叠缓冲液中（25毫摩尔Tris，pH为9.0，0.3 mM的胱氨酸，3.0 mM的半胱氨酸，1mM EDTA中）。德加的复性缓冲液。还要准备600微升10毫米的视黄醇，乙醇在昏暗的红灯。保持在黑暗中冰。的复性缓冲液和维生素A的解决方案都需要新鲜的准备。 </li><li>冷却的蛋白质溶液，至少30分钟的重折叠缓冲液在冰上。较高的温度可能会降低复性RBP的质量。加入1毫升的重折叠缓冲液中滴加和10μl的视黄醇，而蛋白质溶液在冰上在烧杯中剧烈混合溶液中依次。重复此步骤，直到所有的复性缓冲液和维生素A被添加。继续搅拌反应5小时，在黑暗中在冰上。 </li><li>自旋向下折叠反应在24000×g离心30分钟，在4°C。它是必不可少的，以去除聚集体后，复性反应。 </li><li>浓缩上清液使用Amicon Ultra 15集中器（MWCO 10 K）的溶液至约10毫升。不要集中复性的解决方案超过这个水平。集中过多会导致蛋白质聚集和降低最终产量和质量的正确折叠的RBP。稀释浓缩的样品用冷PBS至100 ml。此步骤的目的是在复性的反应干扰镍纯化以删除组件。 </li><li>附带一个更多的时间在该溶液在4℃下24,000 xg离心20分钟重要的是要除去任何沉淀物。将上清转移至两个50ml试管，每片含1毫升的Ni-NTA，已用PBS洗涤浆料。一小时，在4℃下旋转管。孵化稀释后的溶液F或1小时，可能会导致需要被删除的错折叠蛋白的沉淀。 </li><li>旋转管500 xg离心3分钟。仔细收集上清“。任何浮动应该被删除。冷PBS每管30毫升。再次离心，并除去上清液。重复这个过程，直到你什么也看不到，但珠管。 </li><li> Ni-NTA树脂转移到一个空的列。的树脂用20倍柱体积的10mM咪唑，500mM NaCl的在PBS中清洗。洗脱的His-RBP 5倍柱体积的100mM咪唑的PBS中。不要将本溶液在较长的一段时间内，冷冻时间最终的产量和品质。为了最好的结果，净化RBP立即高效液相色谱法。 </li><li>透析的His-RBP从反对的25mM Tris，pH值8.4，和120mM氯化钠的Ni-NTA树脂纯化。甲集中器也可以用于缓冲液交换。离心16,000 xg离心10分钟，在4°C在每次运行前，清除样品的HPLC。 </li><li>使用离子净化全息-RBP HPLC交换柱AX-300通过使用作为在1毫升/分钟的流动相的pH值为8.4的25mM Tris，分步梯度的NaCl（220 mM的12分钟，360毫15分钟，和1,000 mM的15分钟）。随着NaCl浓度的升高，全息-HIS-RBP被释放，而Apo-RBP，错误折叠的RBP留绑定列（ 图1）。如果还没有做最佳RBP重折叠，错误折叠的RBP（ 例如，错误折叠的RBP-I在图1）可以在整个制剂中占主导地位。 </li><li>恢复正确折叠的holo-RBP从高峰分数在360毫米氯化钠。小心地监视正确的折叠和错误折叠的RBP的洗脱曲线。于1000毫米的NaCl，大多数的错误折叠的RBP应该被释放。 </li><li>含有的holo-RBP的馏分被汇集，浓缩，并在4℃下对PBS透析过夜检查的最终产量和质量（330 nm/280纳米）由分光光度计。正确折叠的产品应具有330 nm/280 nm的比率稍微高于1（图 1）。 </li><li>为了生产ËAPO-RBP，加等体积的正庚烷的纯化的holo-RBP，轻轻混匀，旋转过夜后，于4℃离心10分钟，在4℃，并小心地16000克的底部的水相转移到新的试管（避免沉​​淀物的界面处）。重复该过程三次，为每个与孵育3小时。检查，以确保在330 nm处的峰值下降到本底水平上的NanoDrop分光光度计的吸收。 </li></ol> 2。实时监控的STRA6催化视黄醇发行和视黄醇装载的<ol><li>座的黑色96孔显微荧光-2板（Thermo Scientific的）用200μl的拦截器，酪蛋白（Pierce公司）过夜，在4℃，以防止非特异性的holo-RBP粘附到塑料。酪蛋白拦截器的所有阻塞的解决方案，我们测试给出了最好的阻断作用。 </li><li>表达STRA6或控制细胞的细胞从新鲜制备的膜用PBS洗涤，并通过微量注射器（GastiGHT＃1710）的6倍，以产生均匀的悬浮液在PBS中。我们通常使用来自于1/100〜1/20的每个反应的100毫米的培养皿上生长的细胞的膜。 </li><li>洗净一次的显微荧光-2板的包被的孔用200μl的PBS。膜悬浮液（每孔50μl）加入前板置于冰上冷却。 </li><li>实时监控的POLARstar欧米茄（BMG LABTECH）用荧光光学测量视黄醇荧光激发滤光片320ex和发射滤光片460-10。程序设置到端点的阅读模式。板模式也可以被用于测量的时间过程，如果不发生变化的时间间隔。每个时间点的信号是10次测量的平均值（轨道平均具有直径为2毫米）的增益被设定为1800。在500 RPM，使用双轨道的晃动，在每次测量前板振动，持续10秒。 </li><li>要启动的视黄醇的释放试验，井读取一次使用端点读数m颂歌的holo-RBP之前（通常为1最终浓度10μM）0分钟时加入。尽快的holo-RBP被添加时，该反应连续监测每5-10分钟为1-3小时。要启动的视黄醇的的加载测定，全反式视黄醇（通常为1最终浓度10μM）的被添加在昏暗的红色光的孔。读板，一旦，APO-RBP前加0分钟。尽快的apo-RBP被添加时，该反应连续监测每5-10分钟为1-3小时。 </li><li>完成所有的测量后，下载的原始数据（例如，在图2所示）的的欧米加数据分析软件（BMG LABTECH）到Microsoft Excel进行数据分析。每个时间点生成一个文件，其中包含所有的实验条件下的读数。例如，如果有20个时间点，20个文件合并成一个Excel文件进​​行数据分析。 </li><li>进行数据分析，添加前的视黄醇或的holo-RBP 0分钟时的荧光信号被认为是背景信号ð减去最终在所有时间点的荧光信号。虽然背景信号比较低荧光的holo-RBP视黄醇，减去背景消除膜的非特异性光散射的影响，并使得有可能集中在0分钟添加视黄醇的holo-RBP或视黄醇的荧光。 </li></ol> 3。催化STRA6视黄醇运输的实时监控，从全息-RBP CRBP-I <ol><li>视黄醇-EGFP的是一个新的荧光共振转移（FRET），最近已经建立的对41。从全息-RBP的STRA6催化视黄醇运输EGFP-CRBP-I实时监控，测量视黄醇-EGFP的荧光共振能量转移。与，6xHis标签（EGFP-CRBP-Ⅰ）的EGFP-CRBP-I融合蛋白在哺乳动物细胞中产生的中和实验前的Ni-NTA树脂纯化。 EGFP-CRBP-I可以被存储在一个短的时间内与蛋白酶抑制剂的PBS在4℃下另外，融合蛋白I的冰冻在-80°C的小样品。 </li><li>在反应之前，准备被阻止的显微荧光-2板和膜如上所述。洗净一次的显微荧光-2板的包被的孔用200μlPBS中，然后加入的膜悬浮液（每孔50μl）。 </li><li>的实时视黄醇-EGFP荧光共振能量转移的320ex激发滤光片和排放过滤器460-10和510-10使用POLARstar欧米茄同时决斗发射荧光​​光学测量。程序设置到端点的阅读模式。板模式也可以被用于测量的时间过程，如果不发生变化的时间间隔。每个时间点的信号是10次测量的平均值（轨道平均具有直径为2毫米）的增益被设置为在1,800每通道。在500 RPM，使用双轨道的晃动，在每次测量前板振动，持续10秒。 </li><li>为了启动反应，EGFP-CRBP-I（通常为1最终浓度10μM）加入到孔中。读板CE使用端点的阅读模式之前的holo-RBP加0分。尽快的holo-RBP被添加，将反应连续监测通过测量每5-10分钟1-2小时。 </li><li>所有测量完成后，下载从欧米茄数据分析软件中的原始数据（例如， 图3中所示）到Microsoft Excel如上所述进行数据分析。 </li><li>视黄醇-EGFP FRET的计算方法是在比受主/施主发射峰47的动态变化。的等式41来计算这个比例是[（510吨-510）/（-460460吨b）〕，其中460吨510吨，510 B，和460 B代表在510 nm处的排放量，在反应开始后（吨=时间点），视黄醇或前的holo-RBP在510 nm处被添加（β=背景），在460nm处的反应开始后（t =时间点），和视黄醇或的holo-RBP前在460nm处被添加（B =背景）。 </li></ol>

没有利益冲突的声明。

我们在这里分享一个优化的RBP生产协议，因为RBP生产和纯化过程是产生正确折叠RBP的关键。由于错误折叠RBP物种和即使在HPLC纯化的细菌产生的RBP的细菌蛋白质的存在下痕量的可能性，它是有帮助使用本机RBP从血清，确认有关RBP的结论。尿RBP，市场上可买到的，是一种复杂的混合物许多种RBP，包括APO-RBP和全息-RBP 48,49。 这里所描述的实时监测技术基础上的自身荧光的视...

维生素A是一种有机分子，是人类生存和几乎所有的人体器官的正常运作至关重要。维生素A衍生物（视黄醇）参与不同的生物化学和细胞的活动，包括光检测视力1,2和调节胚胎发育过程中的基因表达和蛋白质的翻译和成人组织3-6。虽然维生素A有能力扩散全身，演变与血浆视黄醇结合蛋白，一个特定的载体蛋白，维生素在血液中的运输，以实现高效率和特异性，以避免毒性与随机扩散7-10。高亲和力受体结合的限制性商业惯例，并采取了维生素A的假设在20世纪70年代11-13。尽管RBP受体的存在14-31的三十年中积累的证据，受体假说争论多年的existence的全息-RBP一个不正确的定义。全息-RBP的正确定义是，它是维生素A和RBP高亲和1:1的复合物之间。重复提取的holo-RBP由有机溶剂是必要的产生的apo-RBP。这个定义所使用的几乎所有的实验室研究RBP的7,9,32-35或在RBP受体14-31,36-42。全息-RBP被用来反驳的RBP受体的存在是不正确的定义与APO-RBP的急性的免费视黄醇的混合物。由于RBP受体的功能的维生素A吸收全息-RBP-RBP全息释放视黄醇，RBP受体不发挥作用，在视黄醇摄入维生素A是开始（所提出的不正确的定义全息RBP）。 最近的研究发现RBP受体作为一个multitransmembrane的结构域蛋白称为STRA636和其功能的维生素A吸收从全息-RBP 36-43强烈反对的假设，即RBP不需要的受体提供维生素A的详细分析表明STRA6有9个跨膜结构域与位于细胞外的N-末端和C-末端位于细胞内40。位于跨膜6和图7之间是一个必不可少的RBP结合结构域39。 STRA6耦合都LRAT和CRBP-I中的维生素A吸收全息-RBP，但既不是LRAT也不是CRBP-I是绝对必要的，增强了STRA6活动41。 STRA6的能力，以促进维生素A的holo-RBP释放的关键是维生素A的吸收活动41。通过依靠STRA6释放其视黄醇，维生素A交付RBP可以运输维生素A的外周组织中的靶细胞，以高特异性和高效率。 RBP受体的存在，不仅历史辩论至关重要的holo-RBP的定义和准备说明，但最近三个相关的论文的基础上的holo-RBP定义不同ferent从原来的和正确的定义44-46。第一篇论文使用的holo-RBP定义被用来否决RBP受体的存在，研究RBP受体44。第二次和第三次的论文想出的holo-RBP与三分之一的定义，使得它更可能为视黄醇的研究，以形成适当的配合物与RBP 45,46。这些编写的研究报告3 H-retinol/RBP由混合的holo-RBP（APO-RBP），用3 H-视黄醇。由于该试验中没有3 H-retinol/RBP形成的，并没有除去过量的游离的3 H-视黄醇45,46，它是不测定3 H-视黄醇的吸收来自3 H-retinol/RBP，但是一个免费的的3 H-视黄醇扩散法。它已被证明先前STRA6不增强细胞摄取的免费视黄醇由38或LRAT CRBP-I 41。几乎所有的视黄醇被绑定到血液中的限制性商业惯例和有没有可检测频率Ë视黄醇。 RBP受体的主要功能是促进维生素A的释放维生素A摄取的holo-RBP在全息-RBP 41。如果人工释放或视黄醇是游离形式与45,46开始，RBP受体是不需要的。获得免费的视黄醇扩散法相比，分析的基础上正确处理的holo-RBP说明，正确的准备RBP显着不同的结果，它的功能分析是至关重要的。 RBP可以41从人血清中纯化的，但是该过程是复杂的和产率是低的。另一种方法是在大肠杆菌中产生RBP 大肠杆菌。因为E.大肠杆菌不正确折叠与多于一对RBP二硫键，如哺乳动物的分泌蛋白的能力，它是必不可少的正确折叠的蛋白质再折叠限制性商业惯例和纯化。错误折叠的蛋白质不仅有不同的行为矫正折叠RBP在不同的检测，但也导致蛋白质聚集在储存过程中。出于同样的原因，APO-RBP只生产高品质的holo-RBP。我们在这里介绍一种优化方案，以生产高品质的RBP与视黄醇通过细菌表达，复性和高效液相色谱（HPLC）分离，100％负载。高效液相色谱（HPLC）分离，不仅能消除错误折叠的RBP，但也明显受到细菌污染，可能会导致严重的文物，如果RBP使用的信号转导实验。我们还描述了两个敏感的实时监测技术研究视黄醇的运输STRA6。这两种技术都依赖于高品质的RBP。由于空间的限制，放射性维甲酸和HPLC-维生素A吸收分析的经典方法是没有描述在这里。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>盐酸胍</td><td> EMD </td><td> 5010 </td><td></td></tr><tr><td>胱氨酸</td><td>西格玛</td><td> C8755 </td><td></td></tr><tr><td>半胱氨酸</td><td>西格玛</td><td> C7352 </td><td></td></tr><tr><td> EDTA </td><td>费舍尔</td><td> BP118-500 </td><td></td></tr><tr><td>三</td><td>费舍尔</td><td> 7786-1 </td><td></td></tr><tr><td> DTT </td><td> EMD </td><td> 3860 </td><td></td></tr><tr><td>视黄醇</td><td>西格玛</td><td> R7632 </td><td></td></tr><tr><td>羧苄青霉素</td><td>费舍尔</td><td> BP2648-5 </td><td></td></tr><tr><td> IPTG </td><td> EMD </td><td> 5810 </td><td></td></tr><tr><td> PBS </td><td> EMD </td><td> 6508 </td><td></td></tr><tr><td>氯化钠</td><td>费舍尔</td><td> BP358-10 </td><td></td></tr><tr><td>的Ni-NTA </td><td> Qiagen公司</td><td> 1018244 </td><td></td></tr><tr><td>咪唑</td><td> EMD </td><td> 5720 </td><td></td></tr><tr><td>庚烷</td><td> EMD </td><td> HX0295-1 </td><td></td></tr><tr><td>拦截器酪蛋白</td><td>刺穿</td><td> 37528 </td><td></td></tr><tr><td>的Amicon Ultra 15集中器（MWCO10 K） </td><td>密理博</td><td> UFC901024 </td><td></td></tr><tr><td>显微荧光-2板</td><td>费舍尔</td><td> 14-245-177 </td><td></td></tr><tr><td>汉密尔顿注射器气密＃1710 </td><td>费舍尔</td><td> 14-824-655 </td><td></td></tr></tbody></table>

real-time analyses of retinol transport by the membrane receptor of plasma retinol binding protein

维生素A是必不可少的眼光和几乎所有的人体器官的生长/分化的。血浆视黄醇结合蛋白（RBP）的原则和具体的载体在血液中的维生素A。在这里，我们描述了一个优化的技术，生产和净化的holo-RBP和两个实时监测技术的研究维生素A高亲和力RBP受体STRA6的运输。第一种技术使得有可能产生大量的高品质的holo-RBP（100％-加载与视黄醇），维生素A运送检测。高品质的RBP功能分析是必不可少的，因为错误折叠的RBP版本维生素容易和细菌的污染在RBP准备的产生伪影。实时监控技术，如电的细胞膜传输方面的研究作出了重要的贡献。 RBP受体介导的视黄醇运输实时分析，直到最近。这里所描述的第二个技术是意图分析L-STRA6催化视黄醇释放或装载。第三种方法是STRA6催化视黄醇运输全息-RBP细胞视黄醇结合蛋白I（CRBP-I）的实时分析。这些技术提供了高灵敏度和分辨率，揭示RBP受体的维生素A的吸收机制。

我们目前在这里代表的holo-RBP生产和净化高效液相色谱法（ 图1），视黄醇STRA6催化释放的holo-RBP和维生素A装到APO-RBP（ 图2）和实时分析的实时分析结果STRA6催化视黄醇运输全息-RBP EGFP-CRBP-I（ 图3）。 没有重折叠，RBP在细菌中产生的几乎完全是由于存在许多不正确的二硫键错折叠。因此，复性的配位体的存在下，视黄醇中是极为重要的，在...

Watch this Scientific Journal Video about Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein at JoVE.com

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

在这里，我们描述了一个优化的技术，生产高品质的维生素A / RBP复杂的和实时监测技术研究维生素A运输STRA6，RBP受体。

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

real-time-analyses-retinol-transport-membrane-receptor-plasma-retinol

Research

JoVE Journal

Biology

13.6K 次观看.  University of California, Los Angeles. 在这里，我们描述了一个优化的技术，生产高品质的维生素A / RBP复杂的和实时监测技术研究维生素A运输STRA6，RBP受体。

视频: Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

科研

生物学

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

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, ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

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 ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量：受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...