Only one of your dialysis buffers has a decent (100 mM) NaCl. Maybe you could try higher salt IN COMBINATION with glycerol. There's also NDSBs that stabilise proteins.
Also, could the high immidazole be keeping the protein happy? You could test this by dialysing into the same buffer with a range of immidazole concentrations. You could try other related natural compounds, histidine, histamine, as well to understand this process better. Cheers, Paul Paul Steven Miller (PhD) Postdoctoral Researcher University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN ---- Original message ---- >Date: Thu, 30 Mar 2017 11:32:05 +0530 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Akilandeswari >Gopalan <akilaibt2...@gmail.com>) >Subject: Re: [ccp4bb] protein precipitation reg >To: CCP4BB@JISCMAIL.AC.UK > > Dear all, > I have used the following buffers for purification > and dialysis. this is fyi. > > Lysis buffer: > > 25mM Tris pH 7 or 7.5 or 8 > > 100-500mM NaCl (increase in salt concentration > increased precipitation of the protein in the column > itself) > > 5mM Beta mercaptoethanol > > 0.5% Triton X 100 > > I have tried with other buffers also. > > a. HEPES buffer pH7.5 > > b. Phosphate buffer pH 7.8 > > c. MOPS buffer pH 8 > > > > Wash and Elution Buffer: > > 25mM Tris pH 7 or 7.5 or 8 > > 100-500mM NaCl > > 20 and 30mM Imidazole for wash > > 300mM for elution > > > > > > Dialysis Buffer: > > 1. Tris 25mM pH 7 > > 2. Tris 25mM pH 7.5 > > 3. Tris 25mM pH 8 > > 4. Tris 25mM pH 7.5, 5% glycerol > > 5. Tris 25mM pH 7.5, 10% glycerol > > 6. Tris 25mM pH 7.5, 20% glycerol > > 7. Tris 25mM pH7.5, 50mM NaCl > > 8. Tris 25mM pH7.5, 100mM NaCl > > 9. Tris 25mM pH7.5, 1mM MgCl[2 > > ]10. Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM > L-Glu > > In all these cases the protein precipitates. i have > tried to do buffer exchange also. i can see > precipitate sticking on the walls of the tube during > the process. > > On Thu, Mar 30, 2017 at 10:14 AM, Debanu Das > <debanu....@gmail.com> wrote: > > Hi Akila, > > In addition to what others have asked about the > dialysis buffer, a few > more comments that might help to decide next steps > because the > precipitation (note precipitation and aggregation > are related but not > synonymous) may be due to several different or > related reasons: > > 1) At what stage are you dialyzing? Is it after > SEC? Could your > protein be too concentrated at that point since > your yield is high > leading to some precipitation? How severe is the > loss? > 2) Did you try more purification before dialysis? > 3) Are you removing the detergent in the buffer > you are dialyzing against? > 4) Can you try buffer exchange during > concentration instead of dialysis? > 5) Try increasing your NaCl concentration and > adding 5-10% glycerol to > improve protein solubility. > 6) Did you try cleaving the C-term His-tag before > dialysis? Did you > try N-term His tag? > 7) Do you really need to dialyze? Did you try > assays or > crystallization trials with the purification > buffer? You can run a SEC > column without imidazole to remove that before > crystallization. > 8) PSI/SBKB TargetTrack can be great resource to > look at > expression/purification protocols for similar > proteins: > http://sbkb.org/tt/ > 9) Also look up the Tb Structural Genomics > resource to see if there is > anything on this target or related targets: > http://www.webtb.org/ > > Best, > Debanu > > On Wed, Mar 29, 2017 at 6:38 AM, Akilandeswari > Gopalan > <akilaibt2...@gmail.com> wrote: > > Dear all, > > I am a PhD student doing structural studies on a > few proteins from > > Mycobacterium tuberculosis. The gene encoding > the proteins I work on are > > cloned into pet22b with c terminal His tag. the > proteins are expressing > > well. upon purification I am getting good yield > of protein but during > > dialysis, the proteins precipitate. Kindly > suggest some solutions to avoid > > aggregation. pI of one protein is 9.7 and that > of the other is 5.6 > > I am using 25mM Tris pH 7.5 and 100 mM NaCl > buffer with 5mM > > beta-mercaptoethanol and 0.5% triton x 100 for > lysis, the same buffer with > > 20-30mM imidazole for washing and 300mM > imidazole for eluting the proteins. > > > > Thank you > > Regards > > Akila > > > > -- > > Akilandeswari G > > > > -- > Akilandeswari G > JRF > C/O Dr. Alamelu Raja > National Institute for Research in Tuberculosis > Chetput, Chennai
