A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts.
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 alpha (ERalpha)-DNA interactions, in which nuclear extracts prepared from MCF-7 breast cancer cells were used as the source of ERalpha protein. Following the binding of nuclear extracts to surface-immobilized estrogen response elements, rabbit anti-ERalpha antibody followed by a secondary antibody (goat anti-rabbit IgG) were applied to recognize the bound ERalpha 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 ERalpha 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 ERalpha measured using the two-step antibody approach is in agreement with that measured for recombinant ERalpha protein (using receptor binding signals).
Su
Xiaodi
X
Institute of Materials Research and Engineering, A*STAR, 3 Research Link, Singapore 117602, Singapore. xd-su@imre.a-star.edu.sg
Neo
Siew Jun
SJ
Peh
Wendy Y X
WY
Thomsen
Jane S
JS
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance / Q000379:methods*
D011506:Proteins; D011960:Receptors, Estrogen; D004247:DNA