Re: [ccp4bb] mammalian cell culture on IMAC

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Difficult molecular replacement in R32 with pseudo translation
From: Ethan Merritt merritt {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2009-10-19

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From: William Scott wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2009-10-19

Subject: Re: mammalian cell culture on IMAC
From: "Oganesyan, Vaheh" OganesyanV {- at -} MEDIMMUNE {- dot -} COM
Date: 2009-10-19

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:bradbennett76@gmail.com]
Sent: Thursday, October 08, 2009 4:43 PM
To: Oganesyan, Vaheh
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, Oganesyan, Vaheh
wrote:

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: CCP4BB@JISCMAIL.AC.UK
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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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Difficult molecular replacement in R32 with pseudo translation
From: Ethan Merritt merritt {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2009-10-19

Next message:
Subject: Re: how to improve Rfree?
From: William Scott wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2009-10-19