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Dear Matt, make sure that you don't have slow or even fast formation of unspecific intermolecular disulfides by simple checks at several time points on SDS-PAGE using sample buffer without DTT. Check the content of free thiols and disulfides of your protein over a period of time. If you need easy and solid protocols, just send me a mail. Guenter 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. > -- *********************************** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [log in to unmask] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966