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We have successfully used ion exchange with a very shallow gradient to separate phosphorylated & non-phosphorylated kinases. WRT the question on using commercially available kinases to phosphorylate the protein, this can be used in conjunction with ion exchange purification or SEC. It can be quite helpful if the phosphorylated protein is your protein of interest but is present in only low amounts. I follow the phosphorylation by mass spec to determine how many PO4 states I am getting. Often, you get more PO4 sites than you bargain for. The protein is then purified by ion exchange or SEC chromatography after that. HTH-- annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) [log in to unmask] "Satheesh Kumar Palani Nathan" <[log in to unmask]> Sent by: "CCP4 bulletin board" <[log in to unmask]> 25-Nov-2008 16:09 Please respond to "Satheesh Kumar Palani Nathan" <[log in to unmask]> To [log in to unmask] cc Subject [ccp4bb] purification of phosphorylated proteins-off topic 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 1. ion-exchange 2. 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!