Protein detection by Western blot via coiled-coil interactions

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DOIResolve DOI: http://doi.org/10.1016/j.ab.2009.12.007
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TypeArticle
Journal titleAnalytical biochemistry
Volume399
Issue1
Pages138140; # of pages: 3
SubjectAlkaline phosphatase; Animal; Bio; Biotechnology; Epidermal growth factor; Peptides; Phosphatase; Protein; Surface plasmon resonance
AbstractWe 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.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute
Peer reviewedYes
NRC number52745
NPARC number14223465
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Record identifier08ca95e5-c824-4164-9c0b-116ea2217207
Record created2010-11-05
Record modified2016-05-09
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