3.6.2 Batch Purification
1. Resuspend pellet in 5 ml cold lysis buffer (lysozyme, DNase1,
and PMSF is added immediately before use).
2. Incubate for 1 h at 4
C under gentle agitation.
3. Pellet insoluble cell debris by centrifugation for 1 h at
17,000 Â g at 4
C.
4. Prepare precharged Ni-NTA agarose beads (650 μl/sample) by
washing three times:
(a) Transfer Ni-beads into 15 ml centrifuge tube.
(b) Fill the tube with H 2 O.
(c) Centrifuge for 5 min at 500 Â g at room temperature.
(d) Carefully discard the liquid.
(e) Repeat steps b–d two more times.
(f) Equilibrate the beads with wash buffer by resuspending
them in 10 ml buffer. Centrifuge for 5 min at 500 Â g and
remove the buffer.
5. Add the cleared lysate (from step 3) to the Ni-NTA agarose
beads. Incubate for 1 h at 4
C under gentle agitation.
6. Centrifuge for 5 min at 500 Â g and 4
C.
7. Remove the supernatant, and add 40 ml cold wash buffer. Turn
the tube gently upside down to resuspend the beads.
8. Repeat steps 6 and 7, then centrifuge again 5 min at 500 Â g
and 4
C.
9. Remove the supernatant without disturbing the pelleted beads.
10. Resuspend the beads in 1.2 ml elution buffer.
11. Centrifuge for 5 min at 500 Â g at 4
C.
12. Transfer the first elution into a clean 1.5 ml tube and keep
on ice.
13. Add 1.2 ml elution buffer to Ni-beads.
14. Centrifuge for 5 min at 500 Â g and 4
C.
15. Transfer the second elution into a new tube.
16. Confirm the size and the purity of the protein using
SDS-PAGE.
17. Estimate the concentration using a NanoDrop, or determine
the concentration more accurately using a spectrophotometer
and using the correct extinction coefficient.
18. Store the protein for short term at 4
C or for longer term at
À20
C. For long-term storage, add 15% glycerol and flashfreeze.
52
Susanne Lu ¨ chow et al.
1. Resuspend pellet in 5 ml cold lysis buffer (lysozyme, DNase1,
and PMSF is added immediately before use).
2. Incubate for 1 h at 4
C under gentle agitation.
3. Pellet insoluble cell debris by centrifugation for 1 h at
17,000 Â g at 4
C.
4. Prepare precharged Ni-NTA agarose beads (650 μl/sample) by
washing three times:
(a) Transfer Ni-beads into 15 ml centrifuge tube.
(b) Fill the tube with H 2 O.
(c) Centrifuge for 5 min at 500 Â g at room temperature.
(d) Carefully discard the liquid.
(e) Repeat steps b–d two more times.
(f) Equilibrate the beads with wash buffer by resuspending
them in 10 ml buffer. Centrifuge for 5 min at 500 Â g and
remove the buffer.
5. Add the cleared lysate (from step 3) to the Ni-NTA agarose
beads. Incubate for 1 h at 4
C under gentle agitation.
6. Centrifuge for 5 min at 500 Â g and 4
C.
7. Remove the supernatant, and add 40 ml cold wash buffer. Turn
the tube gently upside down to resuspend the beads.
8. Repeat steps 6 and 7, then centrifuge again 5 min at 500 Â g
and 4
C.
9. Remove the supernatant without disturbing the pelleted beads.
10. Resuspend the beads in 1.2 ml elution buffer.
11. Centrifuge for 5 min at 500 Â g at 4
C.
12. Transfer the first elution into a clean 1.5 ml tube and keep
on ice.
13. Add 1.2 ml elution buffer to Ni-beads.
14. Centrifuge for 5 min at 500 Â g and 4
C.
15. Transfer the second elution into a new tube.
16. Confirm the size and the purity of the protein using
SDS-PAGE.
17. Estimate the concentration using a NanoDrop, or determine
the concentration more accurately using a spectrophotometer
and using the correct extinction coefficient.
18. Store the protein for short term at 4
C or for longer term at
À20
C. For long-term storage, add 15% glycerol and flashfreeze.
52
Susanne Lu ¨ chow et al.