Immuno-precipitation of phosphorylated (Phospho-Tyr) proteins

Rachel Hodge rho at leicester.ac.uk
Thu May 25 06:12:08 EST 2000


Hi
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 ? 
Thanks 
Rachel


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