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Phosphotyrosine western blotting, HELP! - (Jan/19/2013 )

Hello everyone I'm a masters student just about to start lab work. I have to write a grant proposal for my work and I'm having some issue working out how I am do to my lab work.
I spoke with my supervisor before christmas and thought I understood what I was doing but I am havign some issues.

My project is basically analysing the phosphorylation state of a specific protein. I am treating cells with different substrates and then immunoprecipitating this protein out to see what effect these treatments have on the phosphorylation,

My question is: how would I go about IPing this protein then analysing the phosphorylation state on a western blot?
As far as i understand, I would IP the protein with a specific antibody, (spin the lysate and then collect the pellet and resuspend) and then western blot to make sure it is the correct kD. Then I would use a specific phosphotyrosine antibody to see if it is phosphorylated. But how would I do this via western blotting? Would I just add the phospho-tyrosine antibody to the membrane and then use another antibody against the phosphotyrosine antibody to get a band if it is phosphorylated?

Thanks for the help I am very confused and have been trying for a while to find out a protocol online to no avail!

EDIT: Going over my notes I think I would transfer the protein gel to a membrane and then add the anti-phosphotyrosine antibody onto the membrane which would bind if there is Y-P. Would I then have to use a secondary antibody which is linked to a reporter? Thanks!


Yup essentially correct - you would IP (may not be necessary, potentially native protein will work too) the protein with an antibody against that protein. Run the resulting lysate on a gel, transfer, probe with a phospho-specific primary antibody (use a different species antibody than the one used for IP or you will detect IgG bands too) for the particular phosphorylated residue you are looking at, then add a secondary antibody against the species in which the phospho-antibody was raised. For example use a goat anti-rabbit secondary if your phospho-antibody is raised in rabbit. The secondary or primary antibody may be conjugated to a reporter type system, which is commonly HRP or alkaline phosphatase used for chemiluminescence or metabolizing a coloured substrate, but fluorescent conjugates are becoming more common.