Assay establishment with labeled interactors
|To establish the binding assay, a coupleable small molecule with affinity to the protein of interest (target) is a prerequisite. The target is expressed in appropriate cell lines as fusion to a fluorescent protein (e.g. GFP). The interacting compound molecule is labeled covalently to a fluorescent dye that exhibits no crosstalk to the fluorescent protein used for labeling of the target. The expression level and integrity of fusion proteins in the cellular lysates is tested by Western Blotting, and the identity of the labeled compound is assessed by mass spectrometry. Next, the photophysical parameters of each partner are analyzed individually to enable optimization of the measurement conditions.
To obtain the binding curve, the concentration of labeled compound is titrated in multiple steps over a concentration range from 1 nM to 500 nM against a constant concentration of target. Auto- and crosscorrelation analysis yields concentrations of free and bound interactors as well as the concentration of the dually labeled complexes, from which the dissociation constant can easily be calculated. Additionally, the fraction of unspecifically bound compound can be easily assessed. Within this initial project phase, the kinetics of binding of the labeled tool compound to the target are also determined (see kinetics of labeled interactors).