Detection of Protein-DNA Binding in Crude Nuclear Extract Using Biacore Assay

Abstract

Biacore is a system that is extensively used to characterize the interactions between molecules in terms of their binding specificity, affinity, and kinetics. The practical procedures, however, for measurement of protein-DNA association in crude nuclear extract are yet to be defined. In the present study, DNA fragments with the least protein binding activity were identified in database for transcription factors and included in Biacore assay as control, so that the signals from non-specific binding were markedly suppressed. It was known that when analytes were purified transcription factors, the dissociation curves in Biacore sensorgrams showed exponential tendency. Further analysis showed that the interaction between ERα complex from crude nuclear extract and DNA oligos could be fitted to mono- or bi-exponential functions. Discrimination between orders of exponential function was based on the results of several statistical analyses with an average score of more than 95%. As exponential characteristics allow extrapolation of the dissociation, theoretical amount of bound anti-ERα antibodies could thus be evaluated statistically. Our procedures made Biacore a practical technique like Supershift Assay to measure protein-DNA association in crude nuclear extract with reproducible and reliable results.