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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Methods. 2012 Dec 24;59(3):301–315. doi: 10.1016/j.ymeth.2012.12.005

Fig. 2. MST quantifies the TEM1-BLIP interaction in agreement with SPR literature values.

Fig. 2

A) By fitting the change in thermophoretic depletion upon titration of wt-BLIP to a constant amount of wt-TEM1 labeled with the fluorescent dye NT647 to the quadratic solution of the mass action law, a binding constant of KD=3.8±0.8 nM was determined. B) The W112A-mutation in BLIP reduces the affinity to TEM1 to 0.5±0.1 μM. C) W150A-BLIP binds TEM1 with an even lower affinity of KD=1.7±0.4 μM. D) In a reversed assay design, the concentration of the fusion protein Ypet-wt-BLIP was kept constant while titrating in wt-TEM1. In concordance with the binding curve shown in A, a KD of 4.8±1.7 nM was determined (black circles). Mutated R243A-TEM1 showed a lower affinity of KD=0.19±0.05 μM (red triangles). In cell lysate, the KD between Ypet-wt-BLIP and wt-TEM1 was quantified as 10±4 nM, thus demonstrating the applicability of MST for measurements in complex bioliquids. Notably, the sign of the MST signal amplitude is changed in lysate compared to buffer due to differences in pH, ionic strength etc.