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problems with Immunoprecipitation- Western Blotting - Rabbit IgG secondary givi - (Aug/02/2006 )


I am trying to IP-WB Met and phospho-Met

I am able to detect Met using rabbit polyclonal antibody for IP, then for WB I use a mouse monoclonal primary for Met and goat anti-mouse IgG-HRP for secondary. I get one strong large band ~145kDa (Met) then a smaller one ~98kDa (not sure what this one is...)

When I try to detect phosphorylated Met:
- I use the same rabbit polyclonal antibody for IP
- for WB I use a rabbit polyclonal antibody specific for phosphorylated Met as primary
- as a secondary I use goat anti-rabbit IgG-HRP

When I do this I get multiple strong bands (6), with the strongest around where I expect phospho-Met (145kDa). unfortunately I also get the exact same pattern of bands when I omit the primary antibody step!!!

Why am I getting this when the IP step should be purifying the antigen? Is the rabbit secondary antibody cross-reacting with something? Please help! So confused... sad.gif

Any help would be much appreciated...Thanks.



The 145 kDa could be the rabbit polyclonal for IP, that is not well reduced? It would be surprising but I don't have an other explanation yet.
then it would be recognized by the goat anti-rabbit.
you could control that by omitting your sample in your IP.

About purifying by IP :

It depends how you wash your bands. you can easily co-IP proteins that bound your protein of interest, or proteins that bind to the beads.