Detection of Tyrosine phosphorylation

Johan Lennartsson Johan.Lennartsson at LICR.uu.se
Sun Aug 3 02:28:26 EST 1997


Hi

I am currently working with the SCF receptor tyrosine kinase (c-Kit) and I
have a problem with detecting tyrosine phosphorylation after ligand
stimulation. I am also working with the PDGF receptor and there I have no
problem detecting tyrosine phosphorylation. In both cases I use porcine
aortic endothelial cells transfected with the  receptors. I have checked
the expression using Western Blots and it seems to be OK.
Briefly this is the way I try detect tyrosine phosphorylation:
1) I Starve the cells for 24 h in serum free medium.
2) Incubate the cells with 100uM Na3VO4 for 1 h.
3) Stimulate the cells with SCF (100ng/ml) for 5 to 10 minutes. 
4) Cool the cells quickly and lyse the cells in 20mM Tris, 150mM NaCl, 1%
Triton-        X100, 10% glycerol, 1mM PMSF, 1mM Na3VO4 and 1%trasylol.
5) Immunoprecipitate the receptor with polyclonal antibodies.
6) Separate the proteins with SDS-PAGE and do a Western blot using
antibodies against phosphotyrosine.

Can anyone tell me what might be wrong.

//Johan Lennartsson





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