> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Oganesyan, Vaheh
> Sent: Thursday, October 08, 2009 3:15 PM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] mammalian cell culture on IMAC
>
> Dear All,
>
> 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.
>
> Thank you for your time.
>
> Vaheh
>

The trick we used at Genentech (which I'm still using) was for secreted insect cell proteins, but it should work for you as well.  Add 1 mM NiCl2 and 10 mM CaCl2 to your conditioned media, and adjust the pH to 7.2 - 7.5.  For insect cell conditioned media, this produces a fairly heavy precipitate which appears to contain the nickel-chelating factor.  Most of the time (but not always!) your protein of interest will not be precipitated by this step, and you can spin out the precipitate then load the supernatant on to your nickel column with no further processing.

Hope that helps,

Matt


--
Matthew Franklin , Ph.D.
Senior Scientist, ImClone Systems,
a wholly owned subsidiary of Eli Lilly & Company
180 Varick Street, 6th floor
New York, NY 10014
phone:(917)606-4116   fax:(212)645-2054


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