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1. Prepare the sample
2. Prepare buffers
Mix and dilute buffer concentrates:
a) Binding buffer: 10 mM Imidazole, 20 mM Phosphate, 0.5 M NaCl, pH 7.4–7.6
b) Elution buffer: 500 mM Imidazole, 20 mM Phosphate, 0.5 M NaCl, pH 7.4–7.6
3. Prepare the column
Using a syringe:
a) Wash the column with distilled water
b) Load nickel salt solution
c) Wash the column with distilled water
4. Basic purification protocol
Using a syringe:
a) Equilibrate the column with binding buffer
b) Apply the sample
c) Wash with binding buffer
d) Elute histidine-tagged protein with elution buffer
e) Collect the eluate
f) Check the purification on SDS-PAGE and/or Western blotting

If it is the first time to purify protein, elution buffers should be tried as follows, then find the best one.
Elution Buffers: 1) 1 × Phosphate Buffer, 60 mM imidazole, pH 7.4
2) 1 × Phosphate Buffer, 100 mM imidazole, pH 7.4
3) 1 × Phosphate Buffer, 200 mM imidazole, pH 7.4
4) 1 × Phosphate Buffer, 300 mM imidazole, pH 7.4