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142 Charlton and Zachariou
the maximum operating linear velocity allowed by the stationary support (see
Note 4), that is, 300–1000 cm/h for the stated support. Assume a loading of
no more than 1 mg target protein per ml of stationary support (see Note 5).
However, target proteins in ratio volumes of 300:1 cell culture per Ni-NTA have
been successfully loaded by the authors (see Note 6).
6. Wash stationary support with 10 CV of equilibration buffer at the loading linear
velocity or until the A 280 nm reading is at baseline (see Note 7).
7. Elute protein with up to 5 CV of 0.2 M imidazole + 0.5 M NaCl pH 7 at
33% of the recommended maximum linear velocity of the stationary support,
1000 cm/h for Ni-NTA superflow. Samples should be examined on sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) for purity (20).
If these conditions have not been able to effect complete elution, follow the
steps described in Subheading 3.2. If the eluted product is of insufficient purity,
follow the steps described in Subheading 3.3.
8. After elution of the target protein, the column should be regenerated using 3 CV
of 0.2 M EDTA + 0.5 M NaCl pH 8. Washing linear velocity is not critical as
long as it does not exceed the maximum linear velocity of the stationary support.
9. Wash with 10 CV of Milli Q water.
10. Load column with 2 CV of 0.1 M NiNO 3 (see Notes 8 and 9).
11. Wash with 10 CV of Milli Q water.
12. Store column at 4°C.
3.2. Improving Product Recovery Pilot Investigation
1. Carry out steps 1–6, Subheading 3.1.
2. Proceed immediately to resin regeneration (i.e., stripping of Ni 2+ )with3CVof
0.2 M EDTA + 0.5 M NaCl pH 8 (see Note 10). Washing linear velocity is not
critical as long as it does not exceed the maximum linear velocity of the stationary
support.
3. Wash with 10 CV of Milli Q water.
4. Sanitize the column by washing with 5 CV of 1 M NaOH.
5. Wash with 10 CV of Milli Q water.
6. Load column with 2 CV of 0.1 M NiNO 3 (see Notes 8 and 9).
7. Wash with 10 CV of Milli Q water.
8. Store column at 4°C.
9. If the protein was recovered in step 2 proceed with the steps described in
Subheading 3.2.1., if not proceed with the steps described in Subheading 3.2.2.
3.2.1. Improving Product Recovery Where Binding is IMCC-Histidine
Mediated
1. Carry out steps 1–3, Subheading 3.1.
2. Equilibrate the column with 10 CV of 0.1 M imidazole and 0.5 M NaCl pH 7
(see Note 11). Confirm equilibration by measuring pH and conductivity. Continue
equilibration until pH and conductivity of effluent matches equilibration buffer.