光致交联未修改蛋白（PICUP）适用于淀粉样肽
Summary
光致交联未修改的蛋白质（PICUP）允许低聚物在亚稳态的蛋白质混合物的粒度分布的特性。我们展示PICUP应用程序的三个有代表性的淀粉样肽的40 – 42残留的淀粉样β-蛋白和降钙素形式，控制肽生长激素释放因子。
Abstract
淀粉样蛋白装配成有毒的低聚物是蛋白质错误折叠疾病，包括阿尔茨海默氏症，帕金森氏症，亨廷顿氏病，遗传性肌萎缩性脊髓侧索硬化症，与2型糖尿病的发病中的一个开创性的事件。由于这些蛋白质组件的亚稳的性质，它是很难用传统的方法，如电泳，色谱仪，荧光，或动态光散射，定量，以评估他们的齐聚物的粒度分布。淀粉样蛋白的低聚物存在亚稳态的混合物，其中的低聚物分解成单体，同时关联到更大的集会。 PICUP稳定的共价交联PICUP时，分馏的方法，如十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS – PAGE）或体积排阻色谱法（SEC），相结合，提供快照齐聚物的粒度分布，前交叉存在的低聚物人口联。因此，PICUP使亚稳态蛋白人群的可视化和定量分析，可用于监控大会和破译关系之间的序列修改和齐聚<sup> 1</sup>。机械，PICUP涉及光氧化钌<sup> 2 +</sup在一个三（联吡啶）钌（Ⅱ）配合物（RuBpy）茹<sup> 3 +</sup>与电子受体的存在可见光照射。茹<sup> 3 +</sup>是一个强大的电子氧化剂，能够抽象从邻近的蛋白质分子的电子，自由基生成一种蛋白质<sup> 1,2</sup>。自由基是不稳定的，高活性物种，因此，通过各种内和分子间的反应迅速消失。一个激进的可能会利用高能量的未成对电子的反应与另一种蛋白质的形成一个二聚体自由基，随后失去一个氢原子，并形成了一个稳定的，共价键相连的二聚体的单体。通过类似的机制，以形成高阶低聚物与单体或其他二聚体，然后二聚体可进一步反应。 PICUP相对其他照片或化学交联方法的优点<sup> 3,4</sup>包括短期非破坏性的可见光（≤1秒）的曝光，没有必要<i前事实</i>修改本机的序列和长度为零的共价交联。此外，PICUP使交联蛋白在广泛的pH值和温度范围内，包括生理参数。在这里，我们展示了三个淀粉样蛋白40交叉连接 – 和42个氨基酸残基的淀粉样β-蛋白变体（Aβ40和Aβ42的），和降钙素PICUP应用程序，以及控制蛋白，生长激素释放因子（GRF）。
Protocol
Discussion
PICUP最初开发研究稳定的蛋白复合物 2 。该方法应用定量研究亚淀粉样蛋白的组件，包括Aβ 的 10朊病毒疾病相关的PrP SC 11和α-突触核蛋白 12 。 PICUP实验设计时必须考虑的最重要的因素是试剂的化学计量学，激光照射时间，和样品制备过程。前两个问题，可能需要实证的优化，而后者的问题在很大程度上影响实验数据的解释。特别是对于淀粉样蛋白，亚稳低聚物的粒度分布的测定…
Acknowledgements
这项工作得到了拉里L ·希尔布鲁姆基金会，IIRG – 07 – 58334，从老年痴呆症协会，和来自加利福尼亚州公共卫生部07-65798和NIH / NIA的，2005/2E补助AG027818 AG030709。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Dolan-Jenner 200-W incandescent lamp | Outro | Dolan-Jenner Industries | Model 170-D | The heat generated by the lamp does not affect samples for short incubation periods. |
| 35-mm SLR camera body | Tool | Pentax | SP500 model | In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source. |
| Clear, thin walled PCR tubes | Outro | Eppendorf | 951010006 supplied by Fisher L22-003-24 | |
| Glass vials (1.8 mL) | Outro | Kimble Chromatography | 60940A 2, supplied by Fisher 03-340-60 | |
| GRF | Reagent | Bachem | H-3695 | |
| HFIP | Reagent | TCI America | H0424 | Use in a fume hood. |
| Aβ40 and Aβ42 | Reagent | UCLA Biopolymers Laboratory | ||
| Calcitonin | Reagent | American Peptide | 22-1-10 | |
| Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate | Reagent | Sigma | 224758-1G | Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h. |
| Ammonium persulfate | Reagent | Sigma | A-7460 | Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. |
| β-mercaptoethanol | Reagent | Sigma | M7154-25 ML | Can be used when SDS-PAGE analysis is performed. |
| Novex Tricine SDS Sample Buffer (2x) | Reagent | Invitrogen | LC1676 | |
| XCell SureLock Mini-Cell | Tool | Invitrogen | EI0001 | |
| Novex Tricine Gels (10-20%) | Outro | Invitrogen | EC6625B0X | |
| Novex Tricine SDS Running Buffer (10x ) | Invitrogen | LC1675 | ||
| Silver Express Staining Kit | Invitrogen | LC6100 |
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