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 >
