can't you try different volumes: i.e. 360 nl protein solution in very little buffer and salt + 40 nl precipitant. Should give roughly 10 mg/ml after equilibration.
Best wishes Kornelius On Thu, 24 Apr 2008 12:42:42 +0100 Roberto Steiner <[EMAIL PROTECTED]> wrote: > I have a similar problem Matthew. > Just got some NVoy from Novexin in which some claim can help if hydrophobic > patches are the main problem. Will see. > > Regards, > Roberto > > On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote: > > > Dear All, > > > > I have a 50kDa protein that is soluble and monodisperse at up to > > approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). > > > > However, it aggregates (probably both via disulphides and via > > 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ > > ml, even in the presence of several detergents. I don't want to add > > DTT since my protein should have several intramolecular > > disulphides....although I do have 2 free Cysteines, partially > > exposed. I have already tried mutating the Cysteines, with little > > improvement. > > > > Any suggestions for obtaining 5-10mg/ml? > > > > Does anybody have good experiences with usin L-Arg and L-Glu (e.g. > > At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, > > 2004, pages 8933...) > > > > Thanks for any input! > > > > Yours, > > > > Matt > > > > ==================================== > > Matthew J. Bottomley, Ph.D. > > Senior Research Biochemist > > IRBM / MRL-Rome > > ==================================== > > > > Notice: This e-mail message, together with any attachments, > > contains information of Merck & Co., Inc. (One Merck Drive, > > Whitehouse Station, New Jersey, USA 08889), and/or its affiliates > > (which may be known outside the United States as Merck Frosst, > > Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact > > information for affiliates is available at http://www.merck.com/ > > contact/contacts.html) that may be confidential, proprietary > > copyrighted and/or legally privileged. It is intended solely for > > the use of the individual or entity named on this message. If you > > are not the intended recipient, and have received this message in > > error, please notify us immediately by reply e-mail and then delete > > it from your system. > > --- > Dr. Roberto Steiner > Randall Division of Cell and Molecular Biophysics > New Hunt's House > King's College London > Guy's Campus > London, SE1 1UL > Phone +44 (0)20-7848-8216 > Fax +44 (0)20-7848-6435 > e-mail [EMAIL PROTECTED] > > > > ---------------------------------------------- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany [EMAIL PROTECTED] Tel -49 7071 601 323 Fax -49 7071 601 349
