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]
[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
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