Avoiding false negative results in specificity analysis of protein-protein interactions

The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 x K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed. Copyright (C) 2010 John Wiley & Sons, Ltd.