Hi there Satheesh, In:
Activation Mechanism of the MAP Kinase ERK2 by Dual Phosphorylation . Cell , Volume 90 , Issue 5 , Pages 859 - 869 B . Canagarajah , A . Khokhlatchev , M . Cobb , E . Goldsmith (PDB # 2ERK) the structure of phosphorylated ERK II was solved by co-expressing a suitable, constitutively active kinase in E-coli. This yielded a mix of diphospho-ERK and ERK, which was isolated by virtue of the his-tag and then separated by MonoQ. I've actually used the same plasmid/protocol myself and it is straight forwards and works very well. Hope this helps, David 2008/11/25 Satheesh Kumar Palani Nathan <[EMAIL PROTECTED]>: > Dear CCP4BB, > > > > This is an off topic question for CCP4BB, I am posting on behalf of my > friend: > > > > Hi, > > I am trying to find a method to purify/separate phosphorylated protein from > unpshosphorylated proteins. I have two approach in my mind > > > > ion-exchange > Fe columns, I tried this too but limited success. > > > > Has any one used any other method for this? It will be great if you can > point some references in this regard. > > > > Another approach is to use commercially available kinase to phosphorylate > the target protein – any comment on this? > > > > All suggestions are welcome. Thank you for your reply in advance. > > Thanks > > Satheesh > > ________________________________ > Dr. P.Satheesh Kumar > Department of Biochemistry and Biophysics > Texas A&M University, 2128 TAMU > College Station, TX - 77843-2128 > USA. > Ph. 1-979-862-7639 > > ________________________________ > Free up your senses. Experience reality up close on MSN video Try it! -- ============================ David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home AIM ID: dbassophile ============================
