When mammalian cell culture is being loaded to GE HisTrap resin Ni ions
are being stripped off the resin, at least in my hands. Did any of you
have similar experience and if so what kind of work-around was found?
Volume is fairly large (3L) and concentration/dialysis have proven to
cause loss of desired protein.
Please share your positive experience.
I get this with insect cell lysates. The solution: load onto ion exchanger
first - not for purification but to get rid of whatever strips Ni2+. Crude
wash with 50 mM salt (or as high as your protein allows) followed by step
elution with 0.5M salt (very few proteins do not elute at 0.5M) --> load
directly onto Ni-NTA. Solves the stripping problem 100%. (If you use step
elution, make sure to NOT use weak exchangers or you will have pH shift of
2-3 units). If the protein binds to cation-exchangers at pH >7, even such a
crude step results in significant purification in itself. If, for some
reason, ion exchangers are not an option, load onto hydroxyapatite and
elute with phosphate. The downside to HA is that it has a lot less capacity
than ion-exchangers and that it needs frequent repacking.
Good luck,
Dima
