Dear Andy, We published a paper a while back showing that we could use up to 2M urea to prevent unwanted aggregation, and crystallize the sparingly soluble protein (Dines et al. J Struct. Biol. 2007) and then showed that we could solve the structure which was not affected by the presence of the urea (Dines et al. JBC 2008). I am pretty sure that what you suggest will be fine, and even if there is residual urea, it will not affect the crystal structure.
Best wishes, Noam [cid:[email protected]] From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Andrew Lovering Sent: Monday, October 30, 2017 5:29 PM To: [email protected] Subject: [ccp4bb] Mild Denaturation to lose ligand Dear All, I have a protein from which I’d like to lose the co-purifying ligand (Coenzyme A). The protein is his-tagged. The plan here is to bind it to a resin and then run over a mild denaturant solution to encourage ligand loss (it seems that extended dialysis does not achieve this). Our starting protocol might aim at using roughly 1-1.5M Urea. If anyone has trialled this (or a different protocol, say extremely high NaCl) for a similar or different protein:ligand combination then I’d be keen to hear your thoughts. Thanks, Andy
