If EDTA is the issue (as a secret component) then I can see that Mg does not
help, because the Binding(Chelating) constant is
10 orders of magnitude higher for Ni than Mg. Ni or Co could however work to
saturate the EDTA.
pH (too basic) I don't think so because the cells would not react well to
any significant change.
Color changes I cannot see, largely because the loaded medium is orange
anyhow.
Thanks for the responses, more testing....right now: good old fashioned
dialysis into the HisTrap loading buffer....
Thx, BR
-----Original Message-----
From: H. Raaijmakers [mailto:[log in to unmask]]
Sent: Montag, 19. Mai 2014 16:55
To: [log in to unmask]
Cc: [log in to unmask]
Subject: Re: [ccp4bb] HisTrap Trap
Hi Bernhard,
I just stumbled over this patent, where they add cobalt or nickel ions:
http://www.google.com/patents/WO2013082518A1?cl=en
[0086] Supplementing cell culture media, such as CD FortiCHOT and Freestyle
293, with metal ions does prevent column stripping and improve histidine
tagged protein binding during IMAC purification. This procedure works for
multiple different IMAC columns including Ni-NTA, TALONR metal affinity,
POROSR MC, as well as ProBondT Ni-IDA (data not shown) resins. A variety of
metals can be used including nickel, copper, and cobalt at concentrations
ranging from 0.05 - 10 mM. MgCl2, however, did not appear to improve
histidine tagged protein binding to IMAC columns. The optimal concentration
for improving protein binding seems to be around 0.5mM for nickel and
copper. The binding times tested varied from minutes (column
protocol) to 4 hours (batch protocol) and in all cases supplementation with
additional metal ions led to increased protein recoveries during IMAC
purification, thus indicating the efficacy of this novel procedure.
Wished I knew this years ago,
cheers,
Hans
Bernhard Rupp schreef:
> Hi Fellows,
>
>
>
> my lab mates successfully expressed a glycoprotein in CHO cells in
> serum free medium, and
>
> the protein captures nicely on HisTrap Excel 1ml columns (obviously,
> high yield is not my problem.).
>
> We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep
> imidazole gradient. 20mM Imidazole buffer for regeneration.
>
> Works fine (and often.see yield remark).
>
>
>
> Overcome by common crystallographers' greed (nor creed), we switched
> to stable xfected HEK293, and cell free medium Gibco CD 293.
>
> The first run gave high final yields & cheers.
>
> The second run less of either, because the small HisTrap column
> essentially dissolved - the medium collapsed,
>
> Ni leaches out, kaput as kaput goes.
>
> A 3rd run on a similar previously working column lead to the same result.
>
>
>
> Only thing changed was the cells and medium. Same buffers, same
> gradients, same Akta equipment, same lab techs.
>
>
>
> Before I improve the statistics by ruining further columns, has
> anybody experienced such a calamity that might
>
> be blamable on secret media components or similar? There is a
> mysterious 'proprietary dispersant' preventing
>
> cell adhesion quoted..
>
>
>
> Best wishes, BR
>
>
>
> ----------------------------------------------------------------------
> ------
> ------------
>
> Bernhard Rupp
>
> <mailto:[log in to unmask]> [log in to unmask]
>
> <mailto:[log in to unmask]> [log in to unmask]
>
> <http://www.ruppweb.org/> http://www.ruppweb.org/
>
> ----------------------------------------------------------------------
> -
>
>
>
>
>
>
|