Hi Bernhard - Like Zhijie, we have also been using a Tangential Flow Filtration (TFF) system to address this same issue with serum-free 293 Freestyle media.
Ours is the Minimate TFF system from Pall; there is also the Millipore Labscale system, and I’m sure others as well. I’ve done only limited direct testing, but I have seen 2- to 3-fold increased yield from a TFF’ed sample in comparison to a non-TFF'ed sample from the same batch of media. I concentrate the media 10x, then change the buffer by continuous diafiltration with 5-6 volumes of Ni-NTA loading/wash buffer. The advantages of TFF over plain dialysis are that it’s faster and uses less dialysis buffer, and due to the continuous agitation, the membrane is less likely to become clogged. On the downside, the membrane capsules are rather pricey, and although it’s mostly hands-off time, the process can still take quite a while, especially with lower-MWCO membranes. Good luck! Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center http://kong.med.nyu.edu/ On May 19, 2014, at 1:54 PM, Zhijie Li <zhijie...@utoronto.ca<mailto:zhijie...@utoronto.ca>> wrote: Hi Bernhard, I too suspect that it is some kind of metal chelating reagent causing the problem (possibly used in medium for carrying Fe2+, as the free Fe2+ is toxic to cells). One simple test would be to load a liter of the unused medium to the Ni2+ column and see what happens. Do you concentrate your medium before pull-down? If it is the chelating reagent in the medium, then concentrating the medium 10-30x may help a lot (also helps yield, since every affinity binding has a Kd). We do that regularly on tangential flow filter columns. It is a little weird that your first run was OK but the later ones suffered from Ni2+ loss. I wonder if you can try stripping the column with EDTA first and then loading it again with fresh NiCl2, every time before loading the medium. The reason to strip it is that I also worry that some Ni2+ on your column might have been partially replaced by some metal ions from the medium. (Loading 1L medium to a 1mL column does not sound like a great idea to me anyways...) Zhijie From: Bernhard Rupp<mailto:hofkristall...@gmail.com> Sent: Monday, May 19, 2014 10:13 AM To: CCP4BB@JISCMAIL.AC.UK<mailto: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/ ----------------------------------------------------------------------- ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. =================================
