Application Notes

5 Conclusions This study demonstrates that MicroScale Thermophoresis (MST) is a very powerful tool for the analysis of protein- protein interactions in solution. The binding affinities which were determined are in excellent agreement with those previously measured on the ProteOn XPR36 system (Table 1). Interaction K d K d MST (nM) Proteon (nM) WT TEM vs WT BLIP 3.5 +/- 0.6 3.2+/- 0.6 WT TEM vs W112A BLIP 474 +/-76 360+/-60 WT TEM vs W150A BLIP 1750 +/- 220 3800+/-600 Ypet BLIP vs WT TEM 4.7 +/- 0.75 3.5 +/- 0.5 Table 1: Comparison of binding affinities of TEM1 to BLIP as analyzed by MicroScale Thermophoresis or SPR. An important advantage of MST- Technology is the buffer independency. The technology is suited to analyze binding interactions in very complex biological samples, such as cell lysates and blood serum. Since interactions take place in biological fluids, it is important to measure interactions under similar experimental conditions. The use of fluorescence-fusion proteins and MST- technology, as was demonstrated in this application note (Fig. 5), opens new possibilities to analyze binding affinities using in vivo-like conditions. This can be useful in order to improve our understanding of macromolecular interactions in crowded macromolecular environments. Materials and Methods Assay conditions Binding of NT-647-labeled TEM1 to BLIP Protein Labeling: 10 µM TEM1 was labeled using NanoTemper's Protein Labeling Kit RED-NHS (L001, NanoTemper technologies, Germany). Sample Preparation: The concentration of labeled TEM1 protein was kept constant at a concentration of 10 nM. The unlabeled binding partners were titrated in 1:1 dilutions. The highest concentration of BLIP was about 20-fold above the expected K d , which was previously determined by SPR. Samples were diluted in MST optimized buffer (50 mM Tris- HCl buffer, pH 7.6 containing 150 mM NaCl, 10 mM MgCl 2 and 0.05 % Tween-20). For measurements, samples were filled into standard treated capillaries (Cat#K002). Binding of Ypet-BLIP to TEM1 The concentration of Ypet-BLIP protein was kept constant at 15 nM. The unlabeled binding partners were titrated in 1:1 dilutions. The highest concentration of TEM1 was about 20-fold above the expected K d , which was previously determined by SPR. Samples were diluted in MST optimized buffer (50 mM Tris-HCl buffer, pH 7.6 containing 150 mM NaCl, 10 mM MgCl 2 and 0.05 % Tween-20). For measurements, samples were filled into standard treated capillaries (Cat#K002, NanoTemper Technologies, Germany). Binding of Ypet-BLIP to TEM1 in cell lysate Preparation of cell lysate: 2 x 10 7 293T cells were lysed using 500 µl of RIPA-buffer. The cell lysate was centrifuged at 15,000 rpm for 5 minutes in order to remove large aggregates and cell debris. Sample Preparation: TEM1 was titrated in 1:1 dilutions (in MST- buffer). Then Ypet-BLIP was added into 200 µl of 293T cell lysate to a final

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