Ni-NTA agarose affinity media
Plastic gravity flow column with porous polyethylene filters and stopper
Cell pellet containing his-tagged recombinant protein
10% SDS-PAGE gel, power source and appropriate visualization equipment
Rollers (in at 4C cabinet)
Dialysis tubing and clips
VivaSpin columns with appropriate molecular weight cut off
Lysis Buffer pH 8 (250mM NaCl, 25mM Tris, 10mM Imidazole, 5mg/L Lysozyme, 5mg/L DNaseI, Roche Complete Protease Inhibitor)
Wash Buffer 1 pH 8 (250mM NaCl, 25mM Tris, 10mM Imidazole)
Wash Buffer 2 pH 8 (250mM NaCl, 25mM Tris, 50mM Imidazole)
Elution Buffer pH 8 (250mM NaCl, 25mM Tris, 200mM Imidazole)
- Clean affinity media: Ni-NTA agarose can be stored in 20% ethanol to prolong its shelf life, wash away the ethanol by pipetting the Ni-NTA agarose slurry into a plastic gravity flow column and washing with 10 bed volumes of distilled water. Then equilibrate with 5 bed volumes of wash buffer. Cap the column and keep the affinity media in wash buffer 1 until use
- Pre-chill all buffers. Prepare Lysis buffer on the day it is to be used.
- Transfer cell pellet to a closed container with 5ml of pre-chilled lysis buffer per gram of cell pellet. Allow to thaw on rollers at 4°C for 30 minutes.
- Disrupt cells by sonication on ice.
- Separate cell debris from soluble protein by centrifugation at 10,000 rpm for 30 minutes at 4°C. Transfer supernatant to a fresh tube.
- Resuspend the Ni-NTA agarose into a slurry in the wash buffer 1 by pipetting up and down.
- Add 1 ml of Ni-NTA slurry to the lysate supernatant. Note that for large volumes of supernatant, a larger volume of slurry will be needed. We used an excess of 1 ml of slurry for every 50 ml of supernatant. Mix the slurry and lysate on rollers for >30 minutes in rollers at 4°C.
- Load the lysate into a plastic gravity flow column at 4°C and collect flow through. Keep all fractions on ice.
- Wash the resin with 10 bed volumes of wash buffer 1, then wash buffer 2, collect fractions.
- Elute the protein with 5 bed volumes of elution buffer.
- Analyse fractions by SDS-PAGE.
- Transfer fraction containing the recombinant protein to dialysis tubing and dialyse into an excessive volume of the desired buffer at 4°C with gentle stirring. Replace the buffer after 45 minutes, then repeat.
- Concentrate the purified protein using a VivaSpin column according to the manufacturers’ instructions.
- Measure the absorbance at 280 nm of the purified protein, and calculate the final concentration using the proteins’ extinction coefficient. This can be calculated by inputting the proteins’ primary sequence into a tool like ProtParam from ExPASy.
- Add Glycerol to a final concentration of 5% v/v and flash freeze in liquid nitrogen prior to storage at -80°C.