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30 Charlton and Zachariou
9. Elute protein with up to 5 CV of 50 mM imidazole + 0.5 M NaCl pH 7 at 33%
of the recommended maximum linear velocity of the stationary support, 235cm/h
for Chelating Sepharose FF. If this is insufficient to effect elution, imidazole
should be taken up to 0.5 M. If the target molecule is still bound then elution
with 0.5 M imidazole + 0.5 M NaCl at pH 5.5 should be tried (see Note 6).
Samples should be examined on sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) for purity (17).
10. 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
(see Note 7).
11. Wash with 10 CV of Milli Q water.
12. Load column with 2 CV of 0.1 M CuNO 3 (see Notes 8 and 9).
13. Wash with 10 CV of Milli Q water.
14. Store column at 4°C.
3.2. Purification of Proteins Using IMAC Based on Non-Histidine
Selection and High Ionic Strength (see Note 8)
1. Load column with 2 CV of 50 mM metal salt.
2. Wash packed M n+ -IDA column with 2 CV of metal rinsing solution, 50 mM
acetic acid + 0.1 M NaCl pH 4 (see Note 1).
3. Wash column with 5 CV of Milli Q water.
4. Equilibrate packed M n+ -IDA column with 10 CV of 30 mM MES + 30 mM
imidazole + 0.5 M NaCl pH 5.5 or 6 (see Note 10). Confirm equilibration by
measuring pH and conductivity. Continue equilibration until pH and conductivity
of effluent matches equilibration buffer.
5. Load sample containing target molecule that has been pre-equilibrated in equilibration
buffer. As a general rule, loading linear velocities should be between 10
and 33% the maximum operating linear velocity allowed by the stationary support
(see Note 2), that is, 70–235 cm/h for the stated support. Assume a loading of
no more than 1 mg target protein per ml of stationary support (see Note 3).
However, target proteins in ratio volumes of 300:1 cell culture per support have
been successfully loaded by the author (see Note 4).
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 5).
7. Subsequent wash steps can be carried out if deemed necessary (see Table 2). If
a wash step is required follow step 6 with the appropriate wash buffer.
8. Elute protein with up to 5 CV of 30 mM MES + 30 mM imidazole + 0.1 M
K 2 HPO 4 + 0.14 M NaCl pH 5.5 or 6 at 33% of the recommended maximum
linear velocity of the stationary support, 235 cm/h for Chelating Sepharose FF.
If this is insufficient to effect elution, phosphate should be taken up to 0.2 M. If
the target molecule still remains bound, then elute with 30 mM HEPES + 30 mM