phosphorylation - (Apr/18/2005 )
does anybody have an idea of how to phosphorylate membrane fractions. any advice would be appreciated
I have used this approach with significant success
In vitro phosphorylation.
Initially to obtain a membrane fraction use non-denaturing detrgents such as 1% w/v Octyl D glucoside in your lysis buffer.
In vitro phosphorylation was performed as described by Hong et al. (1993). The assay contained 50 mM Tris-HCL (pH 7.4), 50 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 5 uCi [gamma-32P]ATP (AmershamBiosciences), and 10 ug cell free extract. Reactions were incubated at room temperature for 5 minutes, quenched by the addition of 3 X Laemmli sample buffer, boiled, and separated by electrophoresis on a 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and phosphoproteins detected using either autoradiography or a phosphor screen (Molecular Dynamics Model 445 S1).
thanks for the protocol. i also use a similar protocol but i was just interested in knowing with what detergent i should solubilize my protein before i perform the in vitro phosphorylation.
is it possible to do a non-radiolabelled phosphorylation assay and still detect the phosphorylation site by MALDI or ESI-MS?? is it possible to see a mass shift of 80Da.
Yes, you can use non denaturing detergents and still send them for mas spec. analysis. I have used the phosphoprotein purification kit from Qiagen to isolate phosphorylated proteins by IMAC approach. They include a Zwitrinic detergetns in their lysis procedure (CHAPS 1%), I have modified the procedure by adding Octyl D glucoside 1% w/v. this detergent has a high MCM and non ionic, thus non denaturing. I have obtained phosphrylated proteins which I have tested by anti-Phospho-threo and serine antibodies and appears to be phosphorylated and I have sent them for MALDI. The experimental molecular masses of the peptides generated for specfic proteins wehre then analyzed by FindMod on expasy.org against their theoretical counterparts which apparently showed an 80 Da diffrence on the phosphrylated peptide masses. You can use this paper as referernce for this approach
Wilkins, M., Gasteiger, E., Gooley, A., , Herpert, B., Molloy, M., Binz, P-A., Ou, K., Sanchez, J-C., Bairoch, A., Williams, K., and Hochstrasser, D. 1999. High-throughput mass spectrometric discovery of protein post-translational modifications. J. Mol. Biol. 289:645-657.
One point I forgot to mention to you is to further confirm the phosphrylated status of your protein is to treat your lysate with alkline phosphatase, where if the phosphorylated proteins diseappears from the elurtion fract, this would confirm the phosphorylated status of your protein. One drawback of this approach, these columns only purifies thre and ser phosphrylated proteins and not Tyr.
I hope this information is helpfull for you and answered your questions, Good Luck!
its great to know that there is somebody else also who uses the kit from qiagen:D . i have tried using this kit for purified proteins though its only for cell lysates. it seems to work. unfortunately or fortunately(i cant comment now)my protein seems to be already phosphorylated. so i performed the dephosphorylation with ALPase and passed the sample through the column(i removed some resin and prepared a smaller column) and i did get a shift i.e. most of the protein was in the flow thru fraction .
regarding ur comments on MALDI , this procedure never seems to work in my case. have u performed an in-gel digestion with trypsin?? i get so many contaminated peaks that now i have to start working on ESI-MS. i will have to check what is this IMAC approach.
but yes, i will definitely check the reference and also keep in mind ur other suggestions.
IMAc stands for Immoblized Metal affimity chromatography. I have cut my bands from a 10% SDS-PAGE gel and sent them to the Instiute for Molecular BioDesign at the University of Alberta, Edmonton, Canada for the analysis whci they did an ingel digestion with trypsin on the gel peieces I have provided (in gel digestion with trypsin). However, using FindMod to analyze the data helped resolve the matter ( I used the same approach described in the paper I sent you earlier). My results have been submitted in a paper format for Journal of Bactriology for publication last week. I have already presented the data at the Canadian Society of Microbiology 2004 (http://www.csm-scm.org/english/abstracts/public/list_presenter.asp?year=2004) and I had very positive feedback. I hope the best for you as well, good luck!