Fig. 3

Expression and purification of KRas with pCPD and pMCSG7. a-b Expression and solubility of KRas_1–169 and KRas fl. The amount of expressed and soluble proteins were analyzed by SDS-PAGE and measured with ImageJ software. Bars are the average of three independent biological replicates; c Marker (M), Total proteins (Tot), Soluble proteins (Sol), Elution step with 500 mM imidazole containing KRas_1–169 -CPD (El), protein concentration step (C), SEC step (SEC), incubation time with InsP6, 10, 30 and 60 min respectively (10’-30’-60’), Flow-through fraction containing KRas_{1–169 -GAAL} (FT); d SDS-PAGE of KRas_1–169 and KRas_{1–169 –GAAL}; e-f Intrinsic and GAP mediated single-turnover GTP hydrolysis for KRas_1–169 and KRas_{1–169 –GAAL}. GAP-334 was used at two different molar ratios of 1:100 and 1:200 (GAP:KRas). Each curve and bar represent the average of three independent biological replicates