通过卷曲螺旋相互作用进行 Western blot 中的蛋白质检测。

Protein detection by Western blot via coiled-coil interactions.

机构信息

Animal Cell Technology Group, Bioprocess Center, Biotechnology Research Institute, National Research Council Canada, Montréal, Quebec H4P 2R2, Canada.

出版信息

Anal Biochem. 2010 Apr 1;399(1):138-40. doi: 10.1016/j.ab.2009.12.007. Epub 2009 Dec 11.

DOI:10.1016/j.ab.2009.12.007

PMID:20005863

Abstract

We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing. We demonstrated that protein detection with Ecoil-biotin was more specific than with SeAP-Ecoil. We then showed that our approach is as sensitive as conventional detection strategies relying on nickel-nitrilotriacetic acid-horseradish peroxidase (Ni-NTA-HRP), anti-His-HRP, or anti-EGF. Altogether, our results indicate that the E/K coiled-coil system is a good alternative for protein detection by Western blot.

摘要

我们提出了一种通过 Western blot 检测蛋白质的方法，该方法利用了两个从头设计的肽 E 和 K 线圈之间发生的高亲和力相互作用。作为模型系统，用标记有 E 线圈的分泌型碱性磷酸酶 (SeAP)（SeAP-Ecoil）以及生物素化的 E 线圈来揭示 K 线圈标记的表皮生长因子 (EGF)。在这方面，我们首先生产了纯化的 SeAP-Ecoil，并通过表面等离子体共振生物传感验证了其与 K 线圈肽相互作用的能力。我们证明了 Ecoil-生物素的蛋白质检测比使用 SeAP-Ecoil 更具特异性。然后我们表明，我们的方法与传统的检测策略一样灵敏，这些策略依赖于镍-亚氨基三乙酸-辣根过氧化物酶 (Ni-NTA-HRP)、抗 His-HRP 或抗 EGF。总之，我们的结果表明，E/K 卷曲螺旋系统是 Western blot 检测蛋白质的一种很好的替代方法。