Affinity Chromatography - Department of Molecular and Cellular ...

Samples: 2.5 ml cell extract containing expressed GST-(His) 6
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl,
20 mM imidazole, pH 7.4
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl,
500 mM imidazole, pH 7.4
Flow:
2 ml/min, 312 cm/h
Note: No Ni 2+ re-loading of the column between the runs
6
mg eluted GST-(His)
3.0
2.5
2.0
1.5
1.0
0.5
0
1 2 3 4 5 6 7 8 9 10
Run No.
Result:
Run No. Eluted GST-(His) 6 , total mg
1 2.76
2 2.82
3 2.83
4 2.72
5 2.71
6 2.65
7 2.64
8 2.63
9 2.54
10 2.59
Fig. 49. 10 repetitive purifications of GST-(His) 6
without reloading the column with Ni 2+ between the runs.
Although metal leakage is very low, the presence of any free metal in the purified product
can be avoided by connecting an uncharged HiTrap Chelating HP column in series after the
first column and before the protein is eluted. This column will bind any metal ions removing
them from the protein as it passes through the second column.
Scale of operation
To increase capacity use several HiTrap Chelating HP columns (1 ml or 5 ml) in series (note
that back pressure will increase) or, for even larger capacity, pack Chelating Sepharose Fast
Flow into a suitable column (see Appendix 3).
Cleaning
Remove metal ions by washing with 5 column volumes 20 mM sodium phosphate,
0.5 M NaCl, 0.05 M EDTA, pH 7.4.
Remove precipitated proteins by filling the column with 1 M NaOH and incubate for 2 hours.
Wash out dissolved proteins with 5 column volumes of water and a buffer at pH 7.0 until
the pH of the flow-through reaches pH 7.0.
Alternatively wash with a non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 min.
Remove lipid and very hydrophobic proteins by washing with 70% ethanol, or with a
saw-tooth gradient 0%–30%–0% isopropanol/water.
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