Protein detection by Western blot via coiled–coil interactions

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• 作者：Cyril Boucher ; Gilles St-Laurent ; Mario Jolicoeur ; Gregory De Crescenzo ; Yves Durocher
• 刊名：Analytical Biochemistry
• 出版年：2010
• 出版时间：1 April 2010
• 年：2010
• 卷：399
• 期：1
• 页码：138-140
• 全文大小：365 K

文摘

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.