Try taking the detergents out of your solublization buffer (that is if you know it's not a membrane protein) and only extract the buffer soluble portion. If you have a tag try switching the tag to a different terminus.
Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so. --- On Wed, 3/23/11, Mark J van Raaij <mjvanra...@cnb.csic.es> wrote: From: Mark J van Raaij <mjvanra...@cnb.csic.es> Subject: Re: [ccp4bb] protein aggregation To: CCP4BB@JISCMAIL.AC.UK Date: Wednesday, March 23, 2011, 2:03 PM - try limited proteolysis to see if you can chop off a disordered region - consider the fact that, although it purifies nicely, your protein may not be well-folded do you have a biochemical activity test? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 23 Mar 2011, at 18:59, Van Den Berg, Bert wrote: > Try different detergents. > Try 10% or more glycerol. > Try adding ligands (if present/known). > Try varying ionic strength and/or pH. > Try giving more specifics so people on the board may be able to help you > better. > > Bert > > > On 3/23/11 1:51 PM, "gauri misra" <kamga...@gmail.com> wrote: > > The protein purifies nicely there is no problem in that. Just at the last > step when it is concentrated it starts precipitating beyond a concentration > of 1mg/ml. > Already the purification buffers have the detergent. > > On Wed, Mar 23, 2011 at 1:44 PM, Kornelius Zeth > <kornelius.z...@tuebingen.mpg.de> wrote: > > 2 M urea and detergents > > On Wed, 23 Mar 2011 13:41:55 -0400 > gauri misra <kamga...@gmail.com> wrote: > > Hi, > > What are the different methods to prevent protein aggregation while > > concentrating so as to increase the concentration of the protein? > > I have some idea of adding EDTA and charged amino acids like L-Arg and > > L-Glu. > > I would appreciate if the readers share their experiences. > > > > Thanks! > > > > Gauri > > ---------------------------------------------- > Kornelius Zeth > Max Planck Institute for Developmental Biology > Dept. Protein Evolution > Spemannstr. 35 > 72076 Tuebingen, Germany > kornelius.z...@tuebingen.mpg.de > Tel -49 7071 601 323 > Fax -49 7071 601 349 > >
