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!
Edited by PaulMG, 19 January 2013 - 10:18 AM.