unspecific binding of protein a sepharose - immunoprecipitation problem (Aug/29/2006 )
Hi there!
I'm trying to coimmunoprecipitate membrane proteins from animal tissue.
Therefore I preclear my lysate with protein a sepharose beads (about for 1 hour). After that I add my ab for 3 hours and then the beads for 2 hours.
The problem is that when I run this first pellet (from the preclearing step, then wash etc) on SDS PAGE, I detect huge bands with my antibodys. 
Is it possible that my proteins of interest bind unspecifically to the beads? How can I avoid this?
My other problem is that I'm not able to boil my samples prior to loading, because my protein tends to aggregate. Is it enough to rock the IP-Pellet with Laemmli buffer at room temperature for a couple of minutes to release the immunocomplex from the beads?
I'll appreciate your help.
I'm trying to coimmunoprecipitate membrane proteins from animal tissue.
Therefore I preclear my lysate with protein a sepharose beads (about for 1 hour). After that I add my ab for 3 hours and then the beads for 2 hours.
The problem is that when I run this first pellet (from the preclearing step, then wash etc) on SDS PAGE, I detect huge bands with my antibodys.
Is it possible that my proteins of interest bind unspecifically to the beads? How can I avoid this?
My other problem is that I'm not able to boil my samples prior to loading, because my protein tends to aggregate. Is it enough to rock the IP-Pellet with Laemmli buffer at room temperature for a couple of minutes to release the immunocomplex from the beads?
I'll appreciate your help.
Unspecific binding of sepharose has been an often discussed problem especially in HPLC utilizing sepharose-matrix based columns. There are some tricks such as blocking with BSA; I would also check if nothing bleeds from your fresh sepharose beads; maybe hey are coated with stabilizing or blocking agents; large amounts of protein may also cause unspecific binding of antibody, however, in most cases, only faint signals; boiling in Laemmli buffer is sometimes to avoid; enough would be incubation for half an hour at 60°C or an hour at 30°C, RT should also work