Name: a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles
Uploaded: 2012-08-31T12:00:00
Description: Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

这项工作是由加拿大健康研究学会的支持。 CSC是由一个从自然科学和工程研究理事会，加拿大博士后奖学金。 FD是Tier II加拿大研究主席。

<html>整体重建过程重整过程开始用纯化的MalFGK 2复合物中的洗涤剂溶解的磷脂存在下通过混合膜支架蛋白（MSP）。该步骤之后是由吸附剂的聚苯乙烯材料称为生物珠或Amberlite（ 图1），由缓慢除去洗涤剂。的自动装配过程的发生是最有可能的，因为非极性疏水磷脂，的MalFGK 2配合物和表面的两亲性的MSP蛋白之间的相互作用。最终产物是一种盘状的颗粒...

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我们描述了一个简单的程序，重组的麦芽糖转运到奈米圆盘。转运体ATPase活性和与可溶性的结合配偶体的男性的相互作用（ 图3）可以重新创建。成功重组的转运到奈米圆盘开启方式为更多的生物物理和生物化学分析。特别令人感兴趣的是系统的分析MalK ATP酶和麦芽糖运输活动的清洁剂，脂质体和奈米圆盘。 ABC转运过程中改变构象的运输周期，但脂质，这些构象变化的贡献仍有待探讨...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的Amicon Ultra-50K离心式过滤器</td><td>密理博</td><td> UFC805008 </td><td>按照制造商的协议，为正确使用</td></tr><tr><td>生物珠SM-2吸附剂</td><td> Bio-Rad公司</td><td> 152-3920 </td><td></td></tr><tr><td> 大肠杆菌总脂质</td><td>阿凡提极性脂质</td><td> 100500C </td><td>溶解在氯仿中，处理以适合的有机溶剂的</td></tr><tr><td> Ni Sepharose 4B柱HP树脂</td><td> GE医疗集团</td><td> 17-5268-01 </td><td></td></tr><tr><td>磷标准溶液</td><td>Sigma-Aldrich公司</td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE医疗集团</td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表一特定的试剂。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 名称 </td><td> 组成 </td><td> 评论 </td></tr><tr><td> DDM股票</td><td> 10％w / v的DDM </td><td>重悬在MilliQ水和存储在-20°C </td></tr><tr><td> MalFGK 2股票</td><td> 1-2毫克/毫升 的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油 0.03％w / v的DDM </td><td>商店纯化后，在-70℃下</td></tr><tr><td> MSP股票</td><td> 10-15毫克/毫升 的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油</td><td>储存于-70℃的纯化后，在&lt;1毫升的等分试样，并避免过度的冷冻/解冻循环</td></tr><tr><td>磷脂股票</td><td> 5纳米E.大肠杆菌总脂质 0.5％w / v的（10毫摩尔）DDM 的50mM Tris-HCl，pH值7.9; 50mM NaCl的</td><td>在4℃下储存1周</td></tr><tr><td> TS缓冲</td><td>的50mM Tris-HCl，pH值7.9; 50mM NaCl的</td><td>储存于4°C </td></tr><tr><td> TSG10缓冲</td><td>的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油</td><td>储存于4°C </td></tr><tr><td> TSG20缓冲</td><td>的50mM的Tris-HCl，PH8 100mM NaCl的 20％v / v甘油</td><td>储存于4°C </td></tr><tr><td> TSGD缓冲</td><td>的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油 0.03％w / v的DDM </td><td>商店在4°C，并添加之前使用的DDM </td></tr><tr><td colspan="3"> 表二。解决方案的食谱。 </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

nanodisc是一个圆盘状的颗粒（〜10-12纳米大），成一小片的磷脂双分子层的陷阱膜蛋白。该nanodisc的研究，尤其是膜蛋白的配体 - 受体相互作用的上下文中的一个特别有吸引力的选择。该方法率先由Sligar和同事的两亲性质的基础上设计高度α-螺旋支架蛋白载脂蛋白A1来自。支架蛋白的疏水性表面的相互作用与脂肪酰基侧链的脂双层，而极性的区域面对的含水环境。奈米圆盘膜蛋白的分析有显着的优势脂质体，因为颗粒是小的，均匀的和水溶性的。此外，通常保留以可溶性蛋白的生物化学和生物物理的方法可以被应用，并从膜的任一侧。在这种可视化的协议，我们提出了一个一步一步的重组的良好特征在于细菌ABC转运，男性，MalFGK的的2复杂。在盘的形成是一个自组装过程中，依赖于疏水相互作用发生期间逐步除去洗涤剂。我们描述的基本步骤，我们强调的重要性选择正确的蛋白质 - 类脂比，以限制形成的聚集体和较大的多分散性的脂质体样颗粒。简单的质量控制，例如凝胶过滤色谱法，天然凝胶电泳和动态光散射光谱，确保光盘已正确重组。

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

奈米圆盘是小盘状颗粒结合成一小片的磷脂双分子层膜蛋白。我们提供了一个可视化的协议，一步一步注册成立的MalFGK2转运到光盘。

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

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