Our HEK medium has some pH-indicator dye in it, and phenol red (phenolsulfonphtalein) is a good guess. Under the assumption that the same indicator was in the other CHO medium, it did not cause a problem there.
Thx! BR -----Original Message----- From: Oganesyan, Vaheh [mailto:[email protected]] Sent: Montag, 19. Mai 2014 17:38 To: [email protected] Subject: RE: [ccp4bb] HisTrap Trap Orange to pink colored media is a sign of phenol presence which is added to buffer. That phenol is the problem. I was having similar problem and asked not to add it. Everything worked well afterwards. Vaheh Oganesyan www.medimmune.com -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Bernhard Rupp Sent: Monday, May 19, 2014 11:30 AM To: [email protected] Subject: Re: [ccp4bb] HisTrap Trap 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:[email protected]] Sent: Montag, 19. Mai 2014 16:55 To: [email protected] Cc: [email protected] 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:[email protected]> [email protected] > > <mailto:[email protected]> [email protected] > > <http://www.ruppweb.org/> http://www.ruppweb.org/ > > ---------------------------------------------------------------------- > - > > > > > > To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. 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