Extended Data Fig. 2: Purification of P-Rex1 DH-PH-DEP1T4L.
From: Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism
a. MonoS ion-exchange chromatography profile of purified P-Rex1 DH-PH-DEP1T4L. b. Coomassie-stained SDS-PAGE analysis of fractions indicated in (a). Representative SDS-PAGE from two independent purifications. c. Comparative MonoS ion-exchange chromatography profile of purified P-Rex1 DH-PH-DEP1. d. Coomassie-stained SDS-PAGE analysis of fractions indicated in (c). Representative SDS-PAGE from three independent purifications. Profiles suggest that T4L insertion promoted the purification of a more homogenous protein preparation. e. Ion-exchange and f. size exclusion chromatography profiles of the ΔN40P-Rex1 DH-PH-DEP1T4L protein utilised to obtain the final 3.2 Å crystal structure. g. Coomassie-stained SDS-PAGE analysis of ΔN40DH-PH-DEP1T4L purification (1) nickel elution, (2) overnight (o/n) TEV digest, (3) pellet after o/n TEV and phosphatase treatment, (4) supernatant after o/n TEV and phosphatase digest, (5-6) main-peak MonoS fractions, (7-9) main-peak size exclusion chromatography fractions. Representative SDS-PAGE from three independent purifications.
