Figure 1. Overview of the applied work-flow for the characterization of SH2-ADAP interactions.

i) Amplification of Linear Templates encoding different SH2 proteins by two steps of PCR. ii) Synthesis of site-specifically biotinylated proteins. Subsequent immobilization on streptavidin-coated magnetic beads and pull-down with ADAP for validation and filtering of direct interactions. iii) Cloning of selected Linear Templates into the expression vector pIX3.0. iv) High yield synthesis of SH2 proteins without biotin and subsequent K_d value determination using Microscale Thermophoresis.