Why does the protein not bind both anion exchange at Tris buffer pH 8 + 10% glycerol and cation exchange at Mes pH 6 + 10% glycerol? Any suggestion for purification of this tag-free protein.

The protein MW 50 kDa and the calculated pI 6.8. Previous result indicated that it can exist as monomer in low salt and 6-mer in high salt.