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