> -----Original Message----- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > Oganesyan, Vaheh > Sent: Thursday, October 08, 2009 3:15 PM > To: CCP4BB@JISCMAIL.AC.UK > 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 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
