solublization of basic protein - (Jan/04/2008 )
I have overexpressed a DNA binding protein of pI 9.8...After purification i am trying to concentrate it...But i am facing aggregation problem ...the buffr i use is Po4 buffer pH 7.4 with 50mM NaCl....
I have also tried pH range of 4.5 to 5.5...Its still aggregating...can anyone suggest a suitable buffer
Thanks in advance
If your protein pI is 9.8, perhaps a much more alkaline buffer is in order - perhaps Tris or Bicarbonate would work
When you say the pI is 9.8, was that calculated theoretically, or by experiment? Your protein might have a sequence that suggests a high pI, but if the basic residues are buried, the actual pI of the native protein may be significantly lower. I would also consider more salt if your downstream experiments can handle it; try 150 mM (near enough to isotonic). If you still have aggregation issues, try something more chaotropic.
protein with this pI should be fairly soluble. hmm. any reason to suspect 1) SH group mediated aggregation (see if beta-mecaptoethanol helps) or 2) your protein has hydrophobic domain(s)?