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>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