We use 8M urea solubilization buffer for our protein in inclusion bodies
and recommended temperature is 10-15C. but in 8M conc the urea does not
dissolve and is in crystalline form only, will it have any effect on
solubilzation efficiency. Our solubilization time is 1 Hr and after that
we centrifuge and use the supernatant for refolding via dialysis. however
the pellet after centrifugation of solubilzation show presence of our
protein on sds page analysis. what should we do so that the process of
solubilization is complete and our protein is not lost in pellet.
Few things come to mind:
1. There is not much difference betweeen 15 and 20C. 8M urea is certainly
soluble at 20C (in fact, it takes quite a bit of time for it to start
crystallizing even on ice; nucleation problems, apparently).
2. Chances are good that you can use as high temperature as you need to
dissolve. I've come across protocols using >=65C in the presence of 100 mM
bME to ensure complete unfolding of the polypeptide and reduction of
disulfides (seems essential for refolding of some proteins and very
detrimental for others).
3. You may need to increase solubilization solution/inclusion body ratio.
4. Some inclusion bodies simply won't dissolve in 8M urea, period. In those
cases the typical choices are GuHCl at 6-8M or high pH (11-12.5 depending
on protein).
Good luck,
Dima
