6 kDa domain titrated into 20 kDa labelled G protein Slow exchange case 19.0 1:0 119.5 1:0.2 120.0 1:0.5 1205 1:1.0 121.0 21 10.2 10.0 9.8 9.6 94 8.4 Measure or estimate Ko e.g.fluorescence.ITC.Biacore.NMR Minimize.if.limited proteolysis egmionsheeeethaneegneseouabe0enpealue.fe Add the compo ent in e 10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 119.0 119.5 120.0 120.5 121.0 121.5 1:1.0 1:0.5 1:0.2 1:0 6 kDa domain titrated into 20 kDa labelled G protein Slow exchange case •Measure or estimate KD e.g. fluorescence, ITC, Biacore, NMR •Minimize interacting region, if necessary e.g. limited proteolysis •Find conditions where both components are stable and the interaction still occurs (e.g. salt, pH dependence) •Find conditions where the exchange regime is favourable (temperature, field strength) •Add the unlabelled component in excess if necessary to saturate the labelled component (except when trying to observe the unlabelled component)