Bernhard, We have had similar, but not identical issues with some insect cell media, as well as column interference by lipid. If we see this, we tend to run all the material with the protein (cell lysate or media) through step-elution ion exchange (quick on, quick off). While the purification is minimal, the protein is more concentrated and performs better on Ni-columns.
Cheers, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On May 19, 2014, at 10:13 AM, Bernhard Rupp <hofkristall...@gmail.com> wrote: > 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 > b...@ruppweb.org > b...@hofkristallamt.org > http://www.ruppweb.org/ > ----------------------------------------------------------------------- > >
