Glutathione sepharose beads

WT human C-Raf and C-Raf-carboxyl terminus were expressed and purified as previously described (36 (link)). Briefly, full-length human C-Raf or C-Raf-carboxyl terminus with an N-terminal GST tag were expressed in sf9 cells. The proteins were extracted and purified on glutathione sepharose beads (GoldBio). Then the GST tag was cleaved off by thrombin protease. Finally, C-Raf was further purified by size-exclusion chromatography (SEC). GST-C-Raf-amino terminus, GST-C-Raf-CR1, GST-C-Raf-RBD, and GST-C-Raf-CRD were expressed in Escherichia coli BL21 (DE3) cells and purified on glutathione sepharose beads (GoldBio) followed by further purification using SEC. Expression and purification of Fab30 (37 (link)), H-Ras (38 (link)), and WT βarr1 and its variants (39 (link)) have been described previously. Expression and purification of FLAG-M2R with C-terminal sortase ligation consensus sequence (LPETGGH) followed by a 6× His-tag was performed as previously described (34 (link)).

About 10 μM βarr1-8× His was incubated with 30 μM V₂Rpp and 30 μM Fab30 at room temperature for 30 min. About 7 μM GST-C-Raf-RBD or other GST-tagged C-Raf variants were then added to the reaction mixture and incubated for 1 h at room temperature. Subsequently, 10 μl of Glutathione Sepharose beads (GoldBio) were added and incubated for another hour at 4 °C with end-to-end rotation. The beads were collected and washed three times using wash buffer (20 mM Hepes, pH 7.4, 150 mM NaCl). Finally, the proteins were eluted in elution buffer (20 mM Hepes, pH 8.0, 150 mM NaCl, and 20 mM reduced glutathione) and visualized by Western blotting using anti-GST antibody (Cytiva; RPN1236) and anti-βarr1 antibody (Cell Signaling; 30036).