Equilibration of Ni-NTA agarose
50ul beads (100ul suspension) of Ni-NTA agarose in 1.5ml Eppendorf tubes.
Wash with 2 x 1.5ml H2O and 2x 1.5ml lysis buffer.
(washing: mix, spin 3min 3500rpm, discharge supernatant).
Resuspend pellet of 10ml cell culture in 1ml lysis buffer (If the expression level of the his-tagged protein is very low, use 100ml bacterial culture).
Lysis buffer: 50mM TrisHCl/NaPO4 pH8.0; 0.1/0.5M NaCl; 0.02% NaN3 (azide).
If necessary add: a)1mM PMSF or protease inhibitor cocktail 1:200 (cocktail for bacterial
cells #P-8849 from Sigma) [This cocktail contains no EDTA, since EDTA can bind to the Ni-beads and prevent the his-tag binding.]
b)Dnase 100U/ml or 25ug/ml + 5 to 10 mg/ml.(?) Incubate 10min 4C.
c)Lysozime 0.2mg/ml. Incubate 10min 4C.not 37c?
d)ßME up to 15mM for proteins with many Cys.
e)0.1% Triton X-100, NP40 or any other detergent that does not destroy
Further information about the components of lysis buffer can be found here.
Sonicate on ice 3 x 30sec or more if the cells are not completely lysed.
Spin 5min (not 15’?) 13000rpm 4C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet).
Resuspend pellet in another 1ml lysis buffer and keep sample of 40ul for PAGE-SDS: insoluble proteins, or unlysed cells.
Use supernatant for next step. Keep sample of 40ul for PAGE-SDS: soluble proteins
1)Mix supernatant of last step with the equilibrated resin 30 to 60 min at 4C end over end.
2)Spin 3min 3500rpm 4C. Discharge supernatant and keep sample of 40ul for PAGE-SDS: unbound material (this material can be used again in case of overloading).
3)Wash beads with 2x1.5ml washing buffer : 50mM NaPO4
pH8.0; 0.3M NaCl and 0.02% Na azide. Keep sample of 40ul for PAGE-SDS of
4)Elute recombinant protein with 2x100ul elution buffer + 100mM Imidazol and 2x100ul elution buffer + 300mM Imidazol (incubate each time 3 to 5min before spin 3min 3500rpm 4C). Elution buffer: as washing buffer + Imidazol.
Keep sample of 40ul for PAGE-SDS of each elution.
5)Resuspend beads in 50ul H2O + 20ul sample buffer x5. Boil and spin. Keep sample of 40ul for PAGE-SDS: not elute protein.
6)Run on PAGE-SDS: crude supernatant; resuspended pellet;
unbound, washings, elutions, and SDS extracted beads.
If most of the protein remains insoluble after extraction, try to change lysis buffer by adding ßME, glycerol, detergents or more NaCl; or try the denaturating protocol extraction. If only part of it is insoluble, re-extract pellet with more buffer, or use more lysis buffer during extraction, or perform a more intensive sonication, or use the “French Press” instrument instead of sonication.
If protein does not bind to the Ni-NTA resin, purify protein under denaturating conditions, or move tag to the opposite end of the protein. Make sure you don’t have EDTA in your lysis buffer. If the protein is only partially bound, use more resin, or bind for longer time, or try additives as ßME, glycerol detergents or more NaCl.
If the his tagged protein elutes with contaminants, try the alternative protocol with increasing Imidazol concentration. Or reduce amount of Ni-NTA resin. Or try additives as ßME, glycerol detergents or more NaCl in the washing/ elution buffers.
If eluted protein seems to be degraded, try to work all the time at 4oC and use additional protease inhibitors during lysis.
If the his-tagged protein does not elute (but remains
attached to the column) use higher Imidazol concentrations for elution
(up to 1M) or additives or elute under denaturating conditions.
Additives: ßME up to 15mM.; Glycerol up to 50%.;
Detergents that do not destroy the protein activity.; NaCl or KCl up to
Alternative protocol if protein elutes with contamination
Make in parallel purification procedures where you include
10-20-30-40 or 50mM Imidazol in the lysis,binding and washing buffer.
Elute directly with 3x100ul elution buffer + 250mM Imidazol.
Check eluted proteins on PAGE-SDS. Expect lower yields but higher purification by increasing the Imidazol concentration