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 FortiCHO 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, TALON® metal affinity, POROS® MC, as well as ProBond 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:b...@ruppweb.org> b...@ruppweb.org > > <mailto:b...@hofkristallamt.org> b...@hofkristallamt.org > > <http://www.ruppweb.org/> http://www.ruppweb.org/ > > ----------------------------------------------------------------------- > > > > > >
