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Daniel, How long are you inducing for and at what temperature? I ave worked with a protein that crashes out completley when the cells are induced at 37 C, when we induced at RT for 3 hours only about 50% of the protein crashed. Also, if you have leaky expression and get more soluble protein when you don't add IPTG why do you need to induce it? Finally, have you tried soulbilizing the ppt'd protein in the pellet and refolding it? hope this helps Rob > I realize this question isn't directly related to the usage of ccp4i, > however in the past information provided to me on the ccp4bb has been > excellent. > > As such, I was wondering if anyone has had trouble with their SeMet > proteins > becoming insoluble upon IPTG induction. The current protein I am working > with has 'leaky' expression and for the wild-type protein when it is not > induced has more soluble protein than when it is. However, in the case of > the SeMet protein, upon induction with IPTG all SeMet protein becomes > insoluble. I was wondering if anyone could provide some insights for > experimental design as to how I can bypass the induction step or improve > solubility with IPTG induction. > > Thanks so much, > > R. Daniel Toogood > MSc. Student > University of Saskatchewan > Saskatoon, Sask. Canada > -- Robert J.Gruninger B.Sc. Graduate student Department of Chemistry & Biochemistry University of Lethbridge, 4401 University Dr. Lethbridge, AB, Canada, T1K 3M4 Phone:(403)329-2746 Fax:(403)329-2057
