Brad,
It looks like the chelator is a reducing agent added to
media for preservation, I guess.
The "cure" is not found yet. I'm just loading ~300
ml per one 5 ml column and it so far serves me well.
The IEX approach did not work well. I’ve
lost more than with IMAC after 3X dilution (with properly chosen resin and
buffer pH).
Your idea works fine: that’s why I
limit load to 300 ml.
Thank you.
_______
Vaheh
From: Brad Bennett
[mailto:[log in to unmask]]
Sent: Thursday, October 08, 2009
4:43 PM
To:
Subject: Re: [ccp4bb] mammalian
cell culture on IMAC
Hi Vaheh-
Well (gulp) you could dilute your solution say 2-10X with buffer with no salt.
You could try small volumes at first and see how dilute the [salt] must be
before your protein sticks to your IEX column.
So, what is in the media that is stripping off the Ni ions? A chelator like
EDTA? And you're sure it's not something more sinister like stripping AND
reducing the Ni? If the media formulation is proprietary, you may never
definitively know. But I wonder if you could add free resin to a small portion
of your solution, spin it down and run the beads on a gel and detect your
protein by silver stain or immunoblot? If, whatever it is, is just stripping
your resin, then maybe you could "pull down" some of your target
protein. Surely some Ni+ will get chelated but maybe it's worth a shot? Maybe
as long as you have plenty of Ni-NTA, Ni-Sepharose or Talon hanging around, you
could give this a try.
You could try salting it out with saturating ammonium sulfate. Not elegant but
it should work and you could resuspend the pellet in whatever buffer and volume
you wanted.
Any other epitopes/affinity handles on this protein? Like Flag or HA?
HTH-
Brad
On Thu, Oct 8, 2009 at 4:15 PM,
In case of cell lysates this may be a good idea since you can adjust
the salt concentration in your sample. In case of secreted proteins it is
probably not so good since the media contains ~150 mM NaCl. In my case this
salt prevents protein from bindinq to Q column.
Thank you anyway.
________________________________
From: CCP4 bulletin board on behalf of Dima Klenchin
Sent: Thu 10/8/2009 3:54 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] mammalian cell culture on IMAC
>
>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
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