Washing: Load the column with 17% of pump B (Elution Buffer), which is equivalent to ~100 mM Imidazole, until the signal stabilizes.
Elution: Load the column with 48% of pump B (Elution Buffer), which is equivalent to ~250 mM Imidazole, until the signal stabilizes. Start collecting the elution in 8 mL tube fractions immediately after the UV signal increases. After approximately 40 mL, the UV signal will stabilize at a low value. Then load the column with 3 c.v. of 100% of pump B (Elution Buffer), which is equivalent to 500 mM Imidazole, until the signal stabilizes again.