测量肽转运到大Unilamellar囊泡
Summary
该协议的细节肽易位到大量的unilamellar脂质囊泡的定量测量方法。这种方法还提供了关于膜易位率的信息，可用于识别肽，有效地和自发地跨越脂质双层。
Abstract
有一个多肽的积极关注，容易交叉没有细胞膜，细胞膜上的受体1协助。其中许多被称为细胞穿透肽，这是经常为他们的潜力作为药物载体 1-3注意到。此外，有越来越多的利益通过非4,5膜裂解机制，特别是那些跨而不引起细胞裂解细菌细胞膜并杀死细胞与细胞内过程的干扰 6,7抗菌肽。事实上，越来越多的作者指出，细胞穿透和抗菌肽1,8之间的关系。膜移位和肽的结构，其转运能力之间的关系过程中的一个坚定的认识需要有效的，重复性检测易位。一些团体提出的方法来衡量大unilamel易位LAR脂质囊泡（LUVs）9-13。 LUVs作为有用的模式细菌和真核生物的细胞膜和肽 14,15荧光研究中经常使用。在这里，我们描述了我们的第一考虑如抗菌肽magainin和buforin二16,17，松崎和同事开发的方法的应用程序。除了这种方法为我们的协议，我们也提出一个简单的数据分析方法，量化易位使用这种检测能力。这种易位检测的比别人的优点是，它有可能提供膜易位率的信息，不需要另外一个荧光标签，可以改变肽属性18，含色氨酸的肽。简单地说，易位到脂质囊泡的能力是衡量本地色氨酸残基和D之间的福斯特共振能量转移（FRET）的功能与外部相关的蛋白质时，LUV ansyl磷脂膜（图1）。细胞穿透肽裂解，因为他们遇到不羁的胰蛋白酶封装的LUVs，LUV膜和下降的FRET信号解离。在DROP FRET为转位肽观察到的信号是明显高于观察LUVs含有胰蛋白酶和胰蛋白酶抑制剂，或当不自发地穿过脂膜是一种肽暴露在含有胰蛋白酶LUVs相同的肽。这种荧光的变化提供了直接量化，随着时间的推移肽移位。
Protocol
1。制备大Unilamellar脂质囊泡（LUVs） 准备LUVs作为细胞膜模拟检测19。 混合磷脂（POPC，760.10克/摩尔），磷脂（POPG. 770.99克/摩尔），5 dimethylaminonaphthalene – 1 -磺酰磷脂（DNS教皇，994.350克/摩尔）（阿凡提极性脂）在氯仿中溶解在50 ：45:5的比例。由于每个检测需要单独编制的实验和控制LUVs，两个脂质蛋糕小瓶应准备。 0.376毫克总脂质（POPC 0.188毫克，0.169毫克POPG，和0.019毫克DNS教…
Discussion
这里介绍的协议，可用于评估脂质囊泡的内部和外部的肽浓度的相对变化。这些变化与易位的能力。这个协议可以被用来确定细胞穿透肽作为药物载体的潜力。由于细胞穿透肽的兴趣的增长，这将是有趣的，看看如何采用定量的方式开发和使用方法，直接测量易位事件。
在我们的实验室中，我们发现，可以通过仔细监测几个特定方面的实验提高检测的一致性。首先，实验组?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者想感谢埃莉诺弗莱明和杰西卡陈有益的讨论。经费是由国家过敏和传染病研究所（NIH – NIAID的）奖R15AI079685和研究公司科特雷尔科学学院奖。由霍华德休斯医学研究所和斯特利基金，附加学生提供了支持。
Materials
| Name of the reagent | Company | Catalogue number |
| 16:0-18:1 PG | Avanti Polar Lipids | 840457C |
| 1:18 Dansyl PE | Avanti Polar Lipids | 810330C |
| 16:0-18:1 PC | Avanti Polar Lipids | 850457C |
| Porcine trypsin | Sigma | T-0303 |
| Bowman-Birk trypsin/chymotrypsin inhibitor | Sigma | T-9777 |
| Mini-extruder | Avanti Polar Lipids | 610000 |
| Ammonium molybdate (para) | Alfa Aesar | 10811 |
| L-ascorbic acid | Sigma | A1417 |
| Hydrogen Peroxide, 30% solution | Mallinckrodt chemicals | 5240-05 |
| HEPES | Sigma | H-3375 |
| NaCl | Sigma | S-9625 |
| EDTA | Sigma | E5134 |
| NaH2PO4 | Sigma | S-0751 |
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Cite This Article
Spinella, S. A., Nelson, R. B., Elmore, D. E. Measuring Peptide Translocation into Large Unilamellar Vesicles. J. Vis. Exp. (59), e3571, doi:10.3791/3571 (2012).