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/ -----------------------------------------------------------------------
