A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts |
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Authors: | Su Xiaodi Neo Siew Jun Peh Wendy Y X Thomsen Jane S |
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Institution: | a Institute of Materials Research and Engineering, A∗STAR, 3 Research Link, Singapore 117602, Singapore b Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore |
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Abstract: | Surface plasmon resonance (SPR) spectroscopy has emerged as a powerful alternative to conventional biochemistry methods for studying protein-DNA interactions that involve recombinant proteins of known identity. There are, however, limited demonstrations of SPR detection of protein-DNA bindings in crude samples, e.g., cell extracts, where the challenge is to detect and identify specific DNA binding protein(s) among other protein components in a physiological setting. We have developed a two-step antibody approach for an SPR study of estrogen receptor α (ERα)-DNA interactions, in which nuclear extracts prepared from MCF-7 breast cancer cells were used as the source of ERα protein. Following the binding of nuclear extracts to surface-immobilized estrogen response elements, rabbit anti-ERα antibody followed by a secondary antibody (goat anti-rabbit IgG) were applied to recognize the bound ERα and amplify the signals, respectively. Through a series of experiments, we have demonstrated that the magnitude of the binding signals from the secondary antibody reflects the affinity by which ERα binds to different DNA sequences. The detection sensitivity is determined by the amount of nuclear extracts and the concentration of primary antibody used. The sequence specificity of the nuclear ERα measured using the two-step antibody approach is in agreement with that measured for recombinant ERα protein (using receptor binding signals). |
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Keywords: | SPR Protein-DNA interaction Nuclear extract Estrogen receptors Estrogen response elements |
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