Prishant, Remember that concentrating by almost any method is a non-uniform process. In your case, right at the membrane the concentration is much higher than in the surrounding solution. As Chris says, frequent efforts to keep the solution well mixed can prevent precipitation. As you mix, look through the tube towards a light to see if you see schlieren patterns, indicative your protein concentrating at the membrane. In the best case, the protein concentration may shoot up too high at the membrane, which induces precipitation, and mixing more will help.
In the really worst case scenario, your protein may not be very soluble under the conditions you have chosen (i.e., pH, salt, ionic strength). If so, you may need to rethink buffer conditions for concentrating in this manner (E.g., more salt, different buffer pH, chaotropic salts like LiCl, etc.). Good luck and don't despair yet. This happens quite often sometimes. Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected] **************************************************************** On Aug 19, 2014, at 3:55 PM, Chris Fage <[email protected]> wrote: > Hi Prashant, > > I typically stop the centrifuge once in awhile and pipet up/down to prevent > the sample from over-concentrating. Depending on how sensitive the sample is, > you may want to do this once every 10-60 min. > > Hope this helps, > Chris > > > On Tue, Aug 19, 2014 at 1:42 PM, Prashant Deshmukh > <[email protected]> wrote: > Hi, > i am concentrating my protein using centricon filter, but it is precipitated > soon. Please help me solving this problem. > Thanks. > Prashant Deshmukh > Dept. of Biophysics, > NIMHANS, > Bangalore 560 029, > E-mail:[email protected] > Mob.No.: +919620986525 >
