Generation of home-made GFP-Trap® beads for ATPase assays

We first expressed and purified a nanobody directed against GFP (nanoGFP). The plasmid vector pOPINE GFP nanobody was a gift from Brett Collins (Addgene plasmid # 49172) [33]. 500 mL of E. coli BL21 cells harbouring this plasmid were cultured at 37°C in LB media supplemented with ampicillin. When the OD₆₀₀ reached ∼0.6, the temperature was dropped to 20°C, and protein expression was induced with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich) for 20–24 h at 20°C. The cells were spun down, flash-frozen in liquid nitrogen, and stored at −80°C. The cell-pellet was re-suspended in 10 mL of lysis buffer (PBS pH 8, 0.5 M NaCl, 5 mM imidazole, 1 mM PMSF and 10 µg/µL of Lysozyme) and left for 1 h at 4°C with gentle rotation. Cells were broken by sonication, maintaining the cells always on ice. Cell lysate was centrifuged at 20,000 × g for 20 min at 4°C, and the supernatant was mixed with 1 mL of TALON® metal affinity beads (Clontech) previously equilibrated with equilibration buffer (PBS pH 8, 0.5 M NaCl and 5 mM imidazole), and incubated for 1 h at 4°C under gentle rotation. After incubation, the beads were sequentially washed with 20 column-volumes of equilibration buffer, 10 column-volumes of equilibration buffer with 20 mM imidazole, and 10 column-volumes of equilibration buffer with 30 mM imidazole. The nanoGFP was eluted with two steps of 5 column-volumes of equilibration buffer with 150 mM of imidazole, and 5 column-volumes of equilibration buffer with 300 mM imidazole. The elution fractions were analyzed by SDS-PAGE, pooled and concentrated using a Vivaspin® 20,5,000 Mw concentrator (Sartorius). The concentrated protein was dialyzed overnight at 4°C to remove the imidazole, further concentrated and stored at 4°C until use. The purified nanoGFP was then covalently bound to agarose beads. 1 ml of NHS-Activated Sepharose 4 Fast Flow beads (GE Healthcare Life Sciences) was washed with 10–15 column-volumes of 1 mM HCl, and equilibrated with PBS pH 7.5. Then, 1 mg of nanoGFP was added to the beads and left overnight at 4°C under gentle rotation, keeping a nanoGFP to beads volume ratio of approximately 0.5–1. After the overnight reaction, the remaining non-reacted sites were blocked by the addition of 10–15 column-volumes of 0.1 M Tris-HCl pH 8.5 followed by a 4 h incubation with this buffer at 4°C. Finally, the beads were subjected to 3 cycles of two sequential washes of 3 column-volumes of 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5 and 3 column-volumes of 0.1 M Acetate buffer, pH 5.0, 0.5 M NaCl. Finally, the home-made GFP-Trap^® beads were equilibrated in PBS pH 8.0 and stored at 4°C until use.