Figure 2: Protein–protein interactions measured with MST.
From: Protein-binding assays in biological liquids using microscale thermophoresis
To determine the affinity of a binding reaction, a titration series of one binding partner is performed while the fluorescent binding partner is kept at a constant concentration. (a) The binding of fluorescently labelled hIFN-γ to a specific antibody is analysed with MST (black circles). The antibody is titrated from 100 pM to 700 nM. The change in the thermophoretic signal leads to a Kd=10±2 nM. For comparison, the dashed lines show binding curves for 1 and 30 nM. (b) The interaction of the intrinsic fluorescent protein GFP with binders of varying affinities is measured. The GBP shows a high affinity of 2.3±2.1 nM (black circles), which is confirmed by a reference experiment on an Attana quartz crystal microbalance device. To ensure that the measured interaction truly represents specific binding, a GBP mutant (R37A) and non-binding RBP are further analysed with MST. The exchange of the arginine at the binding interface reduces the affinity to 80±38 nM (green squares), while the RBP shows the expected baseline (red triangles). The error bars represent the s.d. of each data point calculated from three independent thermophoresis measurements.
