Microscale thermophoresis assay

The peptides were dissolved in sterile water and diluted into 14 gradients of concentration (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.812, 3.906, 1.953, 0.976, 0.488 and 0.244 nM). The αvβ3 integrin was labeled with Protein Labeling Kit BLUE according to the protocol. The fluorescence-labelled avb3 integrin was mixed with peptides solution in 14 concentration gradients at 1:1 ratio, followed by 30 minutes incubation. The capillaries (Cat#K002, NanoTemper) were used to draw the mixed solutions from the 14 tubes, which were then loaded with the order of declining peptides concentration. Pre-scanning was performed to ensure the fluorescent intensities in the 14 tubes were equal. Subsequently, MST binding curves were measured for 3 times and the Kd values were calculated according to the following formulation: f(c)=unbound + (bound - unbound)/2 * (FluoConc + c + Kd - Sqrt ((FluoConc + c + Kd)^2 - 4 * FluoConc *c) ⁴¹.