Hi, there: If you just want to know the percentage of phosphorylation, I highly recommend phospho-tag gel.
You can buy the phospho-tag, and make gels your self. It's very effective. Shawn Toronto On 2015-01-19, at 1:01 PM, Bert Van-Den-Berg wrote: > Try running a regular SDS page gel w/o boiling your sample. Some P proteins > run differently on gel due to the conformational change that is induced. I > agree that you probably have a mixture in which the P form might be the minor > species. I'd probably try to get the IEX to work; given that the sample > sticks poorly to the column one might expect the -2 introduced by the > phosphate to make a big difference. Did you try loading the IEX column at low > salt (maybe as low as 20 mM or so)? > > Bert > ________________________________________ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Srivastava, > Dhiraj [dhiraj-srivast...@uiowa.edu] > Sent: Monday, January 19, 2015 5:39 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] phosphoprotein crystallization > > 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 >>
