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Be aware that some buffers are temperature sensitive and change pH, if this pH change heads toward the pI of the protein it can crash out. On Tue, Aug 19, 2014 at 6:37 PM, Prince, D Bryan < [log in to unmask]> wrote: > Dear Prashant, > > > I have been working with a protein-protein complex expressed in > mammalian cells, and that complex in very poorly soluble. Even with 500mM > NaCl in the buffer, I cannot concentrate the complex to above 3 mg/mL. I > tried an old school technique and precipitated my protein complex with > ammonium sulphate (~80% saturation) on ice. When I recovered my complex, I > was able to get almost 9mg/mL without any precipitation at all. The sample > still crystallizes, but I believe now that the sulphate ion is > shielding/masking part of the protein better than the NaCl did. Perhaps > this would be worth a try for you as well? > > > Another thing you can try with a weakly soluble protein is to set up > various ratios between the protein and the reservoir solution in your > crystallization drop. > > > One point I have not yet seen mentioned is that some proteins are hyper > sensitive to changes in temperature. In my opinion, any protein sample > should be kept cold, (on ice at the bench) UNLESS you have good reason and > biophysical data to show otherwise. If you are concentrating at room > temperature, try concentrating at 4C if you can, you might be pleasantly > surprised. > > > Good Luck! > > > Bryan Prince > > ------------------------------ > > *Confidentiality Notice: *This message is private and may contain > confidential and proprietary information. If you have received this message > in error, please notify us and remove it from your system and note that you > must not copy, distribute or take any action in reliance on it. Any > unauthorized use or disclosure of the contents of this message is not > permitted and may be unlawful. > > > > ------------------------------ > *From:* CCP4 bulletin board <[log in to unmask]> on behalf of Roger > Rowlett <[log in to unmask]> > *Sent:* Tuesday, August 19, 2014 5:50 PM > *To:* [log in to unmask] > *Subject:* [ccp4bb] > > > Some things to try to increase solubility: > > 1. Move the buffer pH away from the expected pI. Proteins have minimum > solubility near their pI values. > 2. Add solubilizing agents to the solution, e.g., 20-50% glycerol. (This > may alter you crystallization screening strategy) > 3. Include some inert salt in the solution to minimize electrostatic > interactions, e.g. 100-500 mM NaCl. > > Ultimately, your protein just may not be very soluble. That is potentially > OK...it will ppt and maybe xtallize well at low concentration. > > Roger Rowlett > On Aug 19, 2014 1:52 PM, "Prashant Deshmukh" <[log in to unmask]> > 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:[log in to unmask] >> Mob.No.: +919620986525 >> >