Infinite m1000 plate

Fluorescence anisotropy measurements were performed in GF buffer (see buffer contents above) supplemented with BSA at a concentration of 1 mg/ml to prevent unspecific interaction of the peptide with the plate. The fluorescently labelled peptide was kept constant at a concentration of 2 nM in a total volume of 150 mL far below K D . PDZK1 constructs were serially diluted by a factor of 0.66. Samples were prepared in triplicates in black flat-bottom polystyrene NBS 96 -well plates (Greiner, . Anisotropy measurements were conducted in an Infinite M1000 plate reader (TECAN) at 25 C. Excitation wavelength was 470 nm and the emitted light was recorded at 530 nm. The anisotropy measurements were setup in such a way that PDZK1 constructs were titrated in excess compared to the fluorescently labelled peptides assuming that only one peptide is bound to any of the PDZK1 constructs at a time allowing a direct comparison of the affinities for the different constructs. Resulting data were analyzed with a 1:1 binding model using Prism (GraphPad software). The affinities for the PEPT2 peptide of the individual domains account well for the observed affinities in the MUT construct but differ for D14, indicating that the spatial arrangement in the full length context plays an important role in binding.