I use already the HisTrap Excel - they really do not leach under normal treatment. Chelator is a prime suspect, agreed.
-----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Reza Khayat Sent: Montag, 19. Mai 2014 17:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] HisTrap Trap I think the new versions of GE's HisTrap columns can address these problems. Try contacting someone at GE. I think a number of labs have had such problems in the past and the culprit has been believed to be a chelator in the media but never confirmed because the manufacturer of the media is not willing to disclose the contents for the media. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org ---- Original message ---- >Date: Mon, 19 May 2014 14:38:12 +0000 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of "Keller, Jacob" <kell...@janelia.hhmi.org>) >Subject: Re: [ccp4bb] HisTrap Trap >To: CCP4BB@JISCMAIL.AC.UK > > > > 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 > > b...@hofkristallamt.org > > http://www.ruppweb.org/ > > ---------------------------------------------------------- ------------- > > > >
