Escherichia coli bl21 de3 plyss competent cells

Pulldown of active Rab5 was achieved using a GST fusion protein with the Rab5-binding domain (R5BD) of Rabaptin5 (residues 739–862). The pGEX-4 T-2/Rabaptin-5:R5BD plasmid was a gift from Dr Guangpu Li (University of Oklahoma). Fusion proteins were expressed in Escherichia coli BL21(DE3) pLysS-competent cells (Invitrogen) and purified using glutathione Sepharose 4B beads (GE Healthcare) according to the manufacturer’s instructions. GST pulldown was performed as previously described (PMID: 25800849). Cells were lysed in lysis buffer (25 mM HEPES-KOH pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 0.1% NP40, 10% glycerol, 1 mM dithiothreitol, and protease inhibitor cocktail) and incubated with GST-Rabaptin(R5BD) for 30 minutes at 4°C on a rotation mixer and washed in lysis buffer lacking detergent (25 mM HEPES-KOH pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 10% glycerol, 1 mM dithiothreitol, and protease inhibitor cocktail).