The supernatant was mixed with 300 µl of Ni - NTA agarose (Qiagen), and the proteins were allowed to bind for 3 hours, followed by washing with 20 ml of resuspension buffer, and eluted with 500 µl of
elution buffer (20 mM NaHPO4, 300 mM NaCl, 10 % glycerol, 150 mM Imidazole, pH 7.8).
The plate was washed with 1.6 mL of binding buffer and proteins eluted with 0.2 mL of
elution buffer (20 mM sodium phosphate, 0.4 M imidazole, 0.5 M NaCl, pH 7.4).
Usually, 1 ml of
elution buffer requires 50 — 100 μl of saturated phosphate buffer depending on the temperature.
Elute the antibodies with 6 ml
elution buffer and neutralize the solution with saturated phosphate buffer.