The column was washed with 10 volumes of column buffer after which the columns were quickly flushed with 3 column volumes of cleavage buffer (20 mM sodium phosphate, 0.1 mM EDTA, 0.5 M NaCl, 100 mM MESNA, pH 6) and incubated overnight at 4°C.
The columns were washed with buffer PB and buffer PE.
After extensive washing in the same buffer, the column was eluted with lysis buffer H containing 500 mM imidazole concentrations.
After washing with the same column buffer, the column was incubated with Cleavage Buffer (20 mM Tris-HCl, pH 8.5, 500 mM NaCl and 50 mM DTT) at 4°C overnight.
The columns were washed with buffer AW1 and AW2.
Then, 500 μl of inhibitor removal buffer were added to the columns which were centrifuged at 14.000 × g for 1 min. The columns were washed with 500 μl washing buffer for two times with centrifugation at 10.000 × g for 1 min for each run.
After adding 100 µl of M-Wash Buffer to the columns and a centrifugation at 16 000 g for 30 s, 200 µl of M-Desulphonation Buffer were added to the columns and incubated at room temperature (RT) for 20 min. Then, the solution was removed by centrifugation at 16 000 g for 30 s and the columns were washed twice with 200 µl of M-Wash Buffer.
The crude cell lysate from 1 L cell culture was filtered and then loaded onto a 5 mL HisTrap FF crude column (GE Healthcare), which was washed with buffer A. The column was eluted with imidazole (linear gradient of 20 300 m m) over 20 column volumes at a flow rate of 2.5 mL min−1.
The lysate was centrifuged at 15,000 g for 30 min at 4°C, and the supernatant was loaded onto a Ni-NTA Superflow column (GE Healthcare) equilibrated with buffer A. The column was washed with buffer B (buffer A containing 20 mM instead of 10 mM imidazole), buffer C (buffer B containing 0.5% (v/v) tween-20) and buffer D (buffer B containing 0.5% (v/v) ethanol).
It was dialysed against buffer A overnight and loaded on DEAE-cellulose (Merck, Bangalore) column, which was preequilibrated with buffer A. The column was washed with buffer A to remove all unbound proteins, and a linear gradient of 0 0.75 M NaCl-added buffer A was used to elute the bound proteins.
