 |
 |
Dear All, Hope all of you are fine, My target protein (helicase protein, Size= 100 kDa, pI = 7.67) is stable with 500 mM NaCl, as evident from eluted fractions of Ni-NTA column. In the SDS-PAGE, there is a nonspecific band around 25 kDa. So, to remove that band, I tried ion exchange column (Q-column). Binding buffer has 200 mM NaCl and elution buffer has 1M NaCl. Protein is eluted at around 100 mM NaCl but it is showing aggregation after elution with ion exchange column. After Ni-column, I am getting 15 ml of 0.9 g/l protein and finally after Q- column, I am getting 120 μl of 6 mg/ml. After Q-column, I am getting sharp peak and sharp band, no any contamination, so purity is excellent . Kindly suggest me, how to stop the aggregation of protein??? I am looking forward for your kind suggestions. Thanking You *Ni-NTA column buffers* *Binding buffer:* 20 mM Tris-HCl at pH 8.5, 0.5 M NaCl, 10 mM β-mercaptoethanol, 10% glycerol, 5 mM Imidazole * Elution buffer:* 20 mM Tris-HCl at pH 8.5, 0.5 M NaCl, 10 mM β-mercaptoethanol, 10% glycerol, 100 mM Imidazole *Ion exchange column buffers* *Binding buffer:* 20 mM Tris-HCl at pH 8.5, 200 mM NaCl, 10 mM β-mercaptoethanol, 10% glycerol, 0.1 mM PMSF *Elution buffer:* 20 mM Tris-HCl at pH 8.5, 1M NaCl, 10 mM β-mercaptoethanol, 10% glycerol, 0.1 mM PMSF Ranti Dev, PhD student, Republic of Korea E- Mail- [log in to unmask] On Fri, Mar 4, 2016 at 11:49 PM, Gert Vriend <[log in to unmask]> wrote: > I saw the cacodylate discussion and remembered I studied cacodylate in > the PDB some 8 years ago or so. The results are part of a page on > 'things that can be found in PDB files'. It seems likely that many of > these problems were solved by the PDB over the past few years. > > http://swift.cmbi.ru.nl/teach/pdbad/PDBAD_5.html > > Greetings > Gert > Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het > handelsregister onder nummer 41055629. > The Radboud university medical center is listed in the Commercial Register > of the Chamber of Commerce under file number 41055629. >