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 MgCl2 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
