Thank you Prof Lewis. Its serine residue which is getting phosphorylated. the pI of my protein is around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow through or very poorly binding to Q and SP sepharose. does any one have any good suggestion for finding out phosphorylation status of a basic protein with pI around 8.0. So far we were using mass spec on tryptic digest but I need a simple and relatively quick method because its not practical to run mass spec on each and every fractions before pooling them.
Thanks Dhiraj On Jan 19, 2015, at 11:13 AM, Rick Lewis <r.le...@ncl.ac.uk> wrote: > is this a phosphorylation on serine, threonine or tyrosine? if so, they > should be quite stable between ~pH 5 and 8. the MS results are probably not > quantitative, just qualitative, and i would expect that the addition of a net > negative 2 charge from the phosphoryl group should make a difference to your > protein on ion exchange. > > i would run a non-denaturing PAGE, and look for differences in Rf for your > protein. i bet that the phosphorylation has not gone anywhere near > completion, especially since you seem to expect a conformational change to > occur to accommodate the PO3, and this is not observed. if your preps are > clean, you shouldn't need a p'tase inhibitor. > > good luck > > rick > > On 19/01/15 16:54, Dhiraj Srivastava 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 > > -- > R. J. Lewis > Professor of Structural Biology > Institute for Cell and Molecular Biosciences > Faculty of Medical Sciences Tel: +44 (0)191 208 5482 > University of Newcastle Fax: +44 (0)191 208 7424 > Newcastle upon Tyne, NE2 4HH, UK Email: r.le...@ncl.ac.uk > URL: sbl.ncl.ac.uk >
