7. Measure ~1.4 mL Ni-NTA containing resin (600 μL resin per
40 mL lysate). Equilibrate the Ni-NTA containing resin twice
with Buffer A by centrifugation at 500 Â g.
8. Add the equilibrated Ni-NTA containing resin to the protein
sample at a ratio of ~600 μL resin per 40 mL of lysate in Falcon
tubes. Incubate overnight at 4
C with slow rotation end
over end.
9. Centrifuge Falcon tubes for 5 min at 500 Â g. Careful discard
the supernatant and transfer the resin pellet (with a little supernatant) to two 5 mL polypropylene columns (Qiagen) which
are capped at the bottom. Let the resin settle and then remove
the bottom cap to allow most of the resin to flow through,
without letting the resin go dry. Cap the tube at the bottom.
The resin should appear red (see Fig. 2c).
Fig. 2 Stages of MBP::Z-ISO purification. (a) Sonication. Upon sonication, the cells should turn from milky
white (left) to translucent red (right). (b) Membrane pellets recovered after ultracentrifugation. (c) Nickel
affinity chromatography resin. (a) Left to right shows the column resin before and after addition of elution
buffer. (d) Typical MBP::Z-ISO protein sample eluted from the nickel affinity resin shows a reddish color
conferred by the heme
58
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n
40 mL lysate). Equilibrate the Ni-NTA containing resin twice
with Buffer A by centrifugation at 500 Â g.
8. Add the equilibrated Ni-NTA containing resin to the protein
sample at a ratio of ~600 μL resin per 40 mL of lysate in Falcon
tubes. Incubate overnight at 4
C with slow rotation end
over end.
9. Centrifuge Falcon tubes for 5 min at 500 Â g. Careful discard
the supernatant and transfer the resin pellet (with a little supernatant) to two 5 mL polypropylene columns (Qiagen) which
are capped at the bottom. Let the resin settle and then remove
the bottom cap to allow most of the resin to flow through,
without letting the resin go dry. Cap the tube at the bottom.
The resin should appear red (see Fig. 2c).
Fig. 2 Stages of MBP::Z-ISO purification. (a) Sonication. Upon sonication, the cells should turn from milky
white (left) to translucent red (right). (b) Membrane pellets recovered after ultracentrifugation. (c) Nickel
affinity chromatography resin. (a) Left to right shows the column resin before and after addition of elution
buffer. (d) Typical MBP::Z-ISO protein sample eluted from the nickel affinity resin shows a reddish color
conferred by the heme
58
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n