蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的
Summary
此稿件描述了SUMO化修饰和着丝粒蛋白，Ndc10和Ndc80的泛素化的检测，在芽殖酵母 。
Abstract
翻译后修饰（翻译后修饰），例如磷酸化，甲基化，乙酰化，泛素化，和蛋白修饰，调节许多蛋白质的细胞功能。与着丝粒DNA着丝粒关联的蛋白质翻译后修饰调解忠实染色体分离，以维持基因组稳定性。生物化学方法如质谱和Western blot分析最常用于识别翻译后修饰的。这里，一个蛋白质纯化方法进行说明，该技术同时SUMO化的动粒蛋白，Ndc10和Ndc80，在酿酒酵母和泛素化的检测。表达组氨酸-Flag标签SMT3（HF-SMT3）和Myc标签Ndc10或Ndc80构建并用于我们的研究的菌株。对于检测蛋白修饰，我们设计了一个协议，以亲和通过使用镍珠纯化His标签的SUMO化蛋白质并用于Western印迹分析用抗Myc抗体检测SUMO化Ndc10一个第二Ndc80。为了检测泛素化，我们设计了一个协议的Myc标签蛋白免疫沉淀和使用免疫印迹分析与抗泛素的抗体，表明Ndc10和Ndc80被泛素化。我们的结果表明，在带有His标记感兴趣的表位标记的蛋白标记的SMT3应变便于多个翻译后修饰的检测。未来的研究应该允许开发这种技术的鉴定和表征蛋白质的相互作用是依赖于特定的PTM。
Introduction
泛素化和SUMO化允许泛素和小泛素样修饰的缀合（SUMO; SMT3在酿酒酵母 1）到目标蛋白。着丝粒蛋白的翻译后修饰影响其细胞水平和蛋白质 – 蛋白质相互作用中不同的细胞周期阶段，以确保忠实染色体分离。例如，蜂窝Cse4 / CENP-A和外着丝粒蛋白DSN1的水平通过泛素介导的蛋白水解以确保基因组稳定性2-5调节。不正确动粒微管附件失稳要求IPL1 /极光酶b激酶，磷酸化而与Dam1复合Ndc80直接与微管6-8交互。尽管年过七旬动粒蛋白的鉴定，也有极少数的研究，调查这些蛋白质翻译后修饰用， 例如 ，泛素和SUMO的修改。一个主要的限制是为了保护PTM的能力s纯化和定制抗体的缺乏检测翻译后修饰如蛋白修饰，磷酸化，甲基化，以及其他的期间。 SUMO化着丝粒蛋白的表征Ndc10，Cep3，Bir1和Ndc80利用自定义抗体9。此外，Ndc10已经牵涉作为底物泛素化10。人类Hec1（Ndc80在酿酒酵母 ）也底泛素化，通过APC / C-hCdh1 E3连接酶调节11。因此，Ndc10和Ndc80是很好的候选方案的最优化同时检测SUMO化和泛素化的S.酵母 。
为了方便SUMO化的标识，我们构建了表达HF-SMT3和Myc标签Ndc10或Ndc80株。使用表位标签（HF：的His6-标志）的背景最小化由于交叉反应性是在多克隆血清了针对一个候选蛋白质经常观察到。我们设计了一个协议，以亲和纯化HF的SMT3偶联物，然后用商业抗Flag和抗Myc抗体检测sumolyated Ndc10和Ndc80在纯化SMT3制剂的存在。对于泛素化，我们设计了一个修饰免疫协议，保留了Myc基因标记着丝粒蛋白的泛素化和进行Western blot分析与商业的抗泛素的抗体来检测Ndc10和Ndc80的泛素化。
Protocol
Representative Results
Discussion
表位标记，如HA，Myc基因，旗，和GST被广泛用于蛋白质的生化分析。菌株与HF-SMT3和Myc标签动粒蛋白质，如Ndc10和Ndc80的构造，有利于翻译后修饰如SUMO化和泛素化的检测。 HF-SMT3 pull down实验允许SUMO化着丝粒蛋白，Ndc10和Ndc80（ 图1）的检测。亲和纯化方案和Western印迹分析使用抗Flag抗体建立的HF-SMT3和目标蛋白质之间相互作用的特异性，因为没有在无HF-SMT3对照株检测修饰的蛋白质。使用对?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Oliver Kerscher for support and advice and members of the Basrai laboratory for their support and comments on the paper. This work was supported by the National Institutes of Health Intramural Research Program.
Materials
| Glass beads | BioSpec Products | 11079105 | 0.5 mm dia. |
| Mini beadbeater | BioSpec Products | #693 | 8 cell disrupter |
| Ni-NTA superflow | Qiagen | 30430 | 100 ml |
| Protease inhibitor cocktail | Sigma-Aldrich | P8215 | 1 ml |
| Anti-c-Myc agarose affinity gel antbody produced in rabbit | Sigma-Aldrich | A7470 | 1 ml |
| Monoclonal anti-Flag M2 antibody produced in mouse | Sigma-Aldrich | F1804 | Primary antibody, dilution 1:1000 |
| c-Myc antibody (A-14) | Santa Cruz Biotechnology | sc-789 | Primary antibody, dilution 1:5000 |
| Purified mouse antibody monoclonal 9E10 | Covance | MMS-150P | Primary antibody, dilution 1:5000 |
| Ubiquitin (P4G7) monoclonal antibody | Covance | MMS-258R | Primary antibody, dilution 1:1000 |
| ECL Rabbit IgG, HRP-linked whole Ab | GE Healthcare Life Sciences | NA934V | Secondary antibody, dilution 1:5000 |
| ECL Mouse IgG, HRP-Linked Whole Ab | GE Healthcare Life Sciences | NA931V | Secondary antibody, dilution 1:5000 |
| DC protein assay | Bio-Rad | 500-0116 | |
| Nitrocellulose membrane | Novex | LC2001 | 0.45 mm pore size |
| NuPAGE 4-12% Bis-Tris Protein Gels | Novex | NP0321BOX | 1.0 mm, 10 well |
| NuPAGE MES SDS Running Buffer | Novex | NP0002 | 20x |
| NuPAGE Transfer Buffer | Novex | NP0006-1 | 20x |
| SuperSignal West Pico Chemiluminescent Substrate | Thermo Scientific | 34078 | |
| 10x PBS pH7.4 | GIBCO | 70011-044 | |
| Blue sensitive X-Ray film | Dbio | DBOF30003 | |
| Automatic developer | Kodak | M35AX-OMAT | |
| Nocodazole | Sigma-Aldrich | M1404 | 50 mg |
| MG-132 | Selleck Chemicals | S2619 | 25 mg |
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