[Protein-analysis] protein complex precipitation

Vinod Agarkar via proteins%40net.bio.net (by agarkarvi from yahoo.com)
Mon Apr 23 11:38:06 EST 2007


Hi Yogesha,
   
  May be you could try the followings:
   
  1) Gradually  increase salt concentration upto 250mM.
  2) Use non-ionic detergents and keep in mind critical micelle concentration (CMC).
  3) Sometimes very  low concentration of urea works. But be carefull. 
  4) Use on-column cleavage.
  5) Most important is for what purpose you need the protein. May be some 20-30% protein is precipitating.
  6) Think about 6XHIS tag.
  7) If nothing work, think about modification of amino acid sequence or renaturations.
   
  I hope, you will get success somewhere. 
  All the best, Vinod Agarkar.



"YOGESHA. S. D" <yogeshasd from yahoo.co.in> wrote:
  Hi,
I am new in the field. One proein has GST and the other not. I got the protein complex fraction from GST column and got purified on superdex 200 with 20mM Tris pH 8.0 and 50mM NaCl. pI of the complex is 6.54. When I subjected the pooled fraction for GST cleavage with protease at 15 degrees the protein precipitates. Any suggestion to solubilize the ppt or better way cleaving is appreciated. Suggestion of reading material will be very helpful to me.
Thanks 
SDY



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