Bernhard, One other point, after rereading your email. For step-elution ion exchange (quick on, quick off), you can use some very cheap and quite robust media the will resist a lot of stuff. Have them pour their own columns and toss it away when it gets too dirty. You don't have to use the high-end stuff for this step (CM-sephedex, Dowex, CM-cellulose, etc.).
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/ > ----------------------------------------------------------------------- > >
