Immuno-precipitation of phosphorylated (Phospho-Tyr) proteins
wgallin at gpu.srv.ualberta.ca
Thu May 25 10:19:27 EST 2000
Rachel Hodge wrote:
> I am trying to assess the degree of phosphorylation a receptor
> undergoes in response to it's ligand plus various other factors.
> If I apply the ligand to my cells and then extract in the presence of
> sodium orthovanadate and do a western using an antibody against
> phospho-tyrosine I see a band appearing which corresponds to the size
> of my receptor and which doesn't appear in the non ligand- treated
> cell extract. However if I immunoprecipitate the receptor (in the
> presence of orthovanadate) I appear to lose the signal with the
> phospho-tyrosine antibody although there is clearly receptor present
> in the treated and untreated immunoprecipitations.
> So finally to the questions:
> 1. Is it possible that during the immunoprecipitation process (which takes
> around 24 hours at 4C) my receptor is becoming dephosphorylated despite
> the presence of sodium orthovanadate (1 mM) in all the buffers ?
> 2. Is there anything else I can do to protect the phosphoylation state of the
> receptor throughout the immuno-precipitation process ?
It is also possible that your receptor is not being tyrosine phosphorylated, but
rather another protein of the same size has the phosphotyrosine.
To check this you should do your immunoprecipitation with the anti-receptor
antibody, as above, and then run both the precipitate and the material that did
not precipitate with the antibody, and blot with the phosphotyrosine antibody.
If the problem is hydrolysis of the phosphotyrosine, then you'll get no signal in
either lane, if the phosphorylation is on a protein other than the one you
expect, the signal will be in the unprecipitated material.
More information about the Methods