Well, this is of only possible relevance, but in a previous lab, we used sf9 cells/media quite a bit, and there was always an issue similar to this, due to [we thought] ferritin being secreted into the medium, and sucking up the metals. Many, in fact, crystallized ferritin this way by mistake! Is the concentrated flow-through colored, indicating protein-bound metals, or do the metals go through a concentrator, indicating free or small-molecule-complexed metals? What about pH?
What about Gibco's formulation-is it available? The mysterious "dispersant" could easily be either some chelator like EDTA. You could try ITC with Gibco in the cuvette, titrate with metals if you have a machine handy-it's just an hour or so. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Monday, May 19, 2014 10:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HisTrap Trap 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<mailto:b...@ruppweb.org> b...@hofkristallamt.org<mailto:b...@hofkristallamt.org> http://www.ruppweb.org/ -----------------------------------------------------------------------
