Purification of 6X HIS Proteins

Contributed by Ian Chin-Sang, Queens University, ON, Canada

Purification of 6XHIS proteins with cell extraction buffer



Extraction buffer: 10mM imidazole, 500mM NaCl, 50mM NaH2PO4, pH8.0, (Optional components: 0.5mM TCEP, 1x protease inhibitor cocktail -Complete PI EDTA free tablets; Benzonase Nuclease HC,3 µl per 60ml extraction buffer).


  • Spin cells harboring 6XHIS proteins in large bottles in Beckman Centrifuge 6000rpm 15 minutes.
  • Dump supernatant
  • Resuspend in 3ml Cell extraction buffer.
  • Grind protein in mortar and pestle plus liquid Nitrogen. (at least 10 minutes.) until becomes a fine powder.
  • Transfer frozen powder to 15ml conical tube and bring volume up to 10 ml.
  • Transfer approx 1ml to microcentrifuge tube and spin for 15 minutes at full speed 4C.
  • While spinning pre-equilibrate Ni-NTA resin with extraction buffer by washing 3X in extraction buffer and resuspend in 50% slurry.
  • Pool all the supernatants from step 6 into a 15ml conical (save 100ul of supernatant =Load) and add about 300 ul of Ni-NTA resin to the supernatant. Save some of the pellet (= insoluble fraction).
  • Bind 6XHIS protein to resin for 20min to 1 hour room temp. Spin and save 100ul of the supernatant= unbound fraction.
  • Wash resin 5 times with extraction buffer (5ml each time).
  • Elute with low pH elution buffer (1ml each elution). Elute 5 X.

This method is based, with permission, on an original protocol available here.