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IMAC of Histidine-Tagged Fusion Proteins 143
3. To the load sample, containing the target molecule, add imidazole to 0.1 M and
NaCl to 0.5 M. Adjust pH to 7. Load the column at 33% of the maximum operating
linear velocity allowed by the stationary support (see Note 4), that is, 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).
4. 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).
5. Attempt to elute the protein with up to 5 CV of 0.5 M imidazole + 0.5 M NaCl pH
7 at 10% of the recommended maximum linear velocity of the stationary support,
300 cm/h for the stated support. Samples should be examined on SDS-PAGE to
evaluate elution success (see Note 7).
6. If unsuccessful, attempt elution with up to 5 CV of 0.5 M imidazole + 0.5M NaCl
pH 5.5 (see Note 12) at 10% of the recommended maximum linear velocity of the
stationary support, 300 cm/h for the stated support. Samples should be examined
on SDS–PAGE to evaluate elution success (see Note 7).
7. If unsuccessful, attempt elution with up to 5 CV of 0.5 M imidazole + 0.5 M
NaCl + 50 mM sodium acetate, pH 4 (see Note 13) at 10% of the recommended
maximum linear velocity of the stationary support. Samples should be examined
on SDS–PAGE to evaluate elution success (see Note 7).
8. If still unsuccessful, repeat the steps described in Subheadings 3.1 and 3.2 with a
different metal ion (see Note 14).
9. Carry out steps 9–12, Subheading 3.1. Substitute metal ions where appropriate.
3.2.2. Improving Product Recovery Where Binding is Non-Specific
1. Carry out steps 1–7, Subheading 3.1.
2. Select a factor from the Table 2 and incorporate it into the elution buffer
(step 7, Subheading 3.1.). Repeat step 7, Subheading 3.1., iteratively with the
factors presented in the Table until protein liberation is detected. Author’s recommendation:
Commence with the most extreme conditions that the target protein
can endure and then work backwards toward milder conditions.
3. Regenerate resin (i.e., strip Ni 2+ ) with 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.
4. Carry out steps 3–8, Subheading 3.2.
5. Include the optimum condition determined in step 2, Subheading 3.2.2., into the
equilibration buffer, load sample and elution buffer and repeat the steps described
in Subheading 3.1 with these modifications (see Note 15).
3.3. Improving Product Purity
1. Carry out steps 1–4, Subheading 3.1.
2. To the load sample containing the target molecule add imidazole to 20 mM and
NaCl to 50 mM. Adjust pH to 7. Load the column at 33% of the maximum