Purification of 6X HIS Proteins

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.

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