Hi Peter, we had a four similar cases recently. What really helped us was to add urea in amounts of 1-2 M. We tested the possible influence on secondary structure by limited proteolysis and CD/Trp-fluorescence and it seemed the protein maintained the same overall conformation as in the absence of urea. This compound also stabilized proteins for crystallization and allowed nicely diffracting crystals in three cases while in the absence we had nothing than precipitate etc.
Good luck Kornelius On Thu, 22 Apr 2010 10:49:38 +0530 peter hudson <peter.hudson.pe...@gmail.com> wrote: > Hello all > > I apolozize for a off topic question on BB. > > I am working with small proetin-protein complex, each have molecular weight > 10kDa. I elute this N-terminal His-tagged complex through Ni-NTA resin in > 50mM of NaH2Po4, 0.3M of NaCl, 5% glycerol and 250mM of Imidazole.Similarly, > Lysis buffer and wash buffers contains same component without and with 20mM > of imidazole respectively. His tag is present on the N-terminus of one > partner of the complex. I am able to purify this protein in small scale and > eluted fraction contains 1-2mg/ml of protein complex. But, when i scale up > the purification volume to 1-2 lit of culture i get a really good yield like > 1or 2 fraction will have 10mg/ml of proetin complex.I store the eluted > fraction both at 4 degree as well as 25 degree. The fraction contains > higher amount proteins get precipitated and settles on the bootom of > microfuge tube while the fractions with less amount of proetin(1-2mg/ml) > remain soluble. can anyone suggest any way to keep this protein complex > solube without breaking the complex? Whether there is any other way to > screen the solubility of protein with different buffers without using > dynamic light scattrer? please suggest any reference which suggest protocols > to check the solubility. I would appreciate the help. > > Thanks in advance > > Peter ---------------------------------------------- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
