an effective modification to your protein preparations would be to add phosphatase inhibitor tablets during all stages of purification. Pierce sell a combination protease and PI tablet that is effective and economical to be added during protein preps. Also you invitro buffers should have inhibitors http://www.piercenet.com/product/pierce-protease-phosphatase-inhibitor-tablets P
On Mon, Jan 19, 2015 at 8:54 AM, Dhiraj Srivastava < dhiraj-srivast...@uiowa.edu> wrote: > I am trying to crystallize a protein that I am phosphorylating in-vitro. > There is no way that I can purify the phosphorylated protein from > unphosphorylated one. I tried Ion exchange and gel filtration for > separating them and they are not working. > Mass spec result shows the phosphorylation at desired site but in the > crystal structure, I am not seeing density for phosphoryl group. I haven't > use any phosphatase inhibitor during crystallization. is it possible that > phosphoryl group is getting lost due to radiation damage or due to > phosphatases, my protein is getting dephosphorylated before making crystal? > I typically get crystals within 24 hours. if I am losing phosphoryl group > due to radiation damage, should I still expect to see conformational > changes due to phosphorylation? in phosphorylated protein, an alpha helix > has to move to accommodate phosphoryl group but I am not seeing any change > in that alpha helix. did any one tried to set crystal tray with > phosphorylation reaction mix without further purification? > > thank you for suggestion. > > Dhiraj > -- P
