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At 10:35 PM -0600 12/2/09, Dima Klenchin wrote: >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). I wouldn't recommend using hot 8 M urea to dissolve proteins that you plan to re-fold for functional studies or crystallization since heating the urea solution will produce a significant amount of isocyanic acid which will react with primary amines in your protein (including the N-terminus) and produce carbamylation modifications (-NH2 to -NHCONH2). If it is that tough to dissolve your inclusion body, I would use 6 M guanidine HCl (in ~200 mM Tris-HCl pH 8.5, 1 mM EDTA, 20-100 mM DTT). You can make 8M urea stocks by dissolving at room temperature (20-25 deg C) but you need to be patient (don't heat). Include an amine-containing buffer like Tris in your final solubilization solution (to help scavenge isocyanate), and don't use old urea stocks since the dissolved urea will decompose into ammonia and isocyanate with time. - John --