The PI of my protein is 7.75, I used 25mM Tris_HCL, 500mM Nacl, 500mM Immidazole, pH 8.75. Till here it is ok. But when I am doing dialysis, always it is aggregated. Till Now I used dialysis buffer containing 25 mM Tris-HCL, 300 mM Nacl, pH 5.75 and also 8. But within one hour it is aggregated.
Which dialysis buffer will be ideal?
Hi there,
My first suggestion would be to repeat the dialysis versus an elution buffer only lacking imidazole (ie. 25mM Tris-HCl, 500mM NaCl, pH 8.75). Ionic strength level might be important for stability. The problem with the dialysis buffer you used is that it lowers NaCl concentration and it lowers pH below the pI of the protein in one case (which might promote precipitation). pH8 is too close to protein pI. It is usually accepted that pH buffer have to be set at least 1 unit of pH away from protein pI to avoid precipitation due to the lack of net charge on the protein.
I agree to try using a dialysis exchange that only removes imidazole first. Many proteins will aggregate and precipitate when slowing going through pI as the pH adjusts so you might need to use another method to adjust pH. What buffer you use will also depend on what you are doing with the protein next such as an additional purification step.
- Badges
- Science method