immunoprecipitation - immunoprecipitation help needed (Oct/03/2010 )
I am currently doing IP for our novel proetin with custom made polyclonal antibody. This antibody works excellent in immunostaining and western blotting. it shows very specific staining and a specific band on WB. Our protein so far seems to be abundantly expressed in mouse tissues and expect that it should IP easily. But somehow I have not been able to immunoprecipitate it. The size of my protein is 22kda.
I am also using baculovirus insect cell system to produce the recombinant protein and I have used the elution fraction after purifying the protein from insect cells for IP but could not get anything. I have followed the protocol from ABCAM (without crosslinking the beads to the antibody step). I use sepharose A for this purpose(I swell and block them myself). I have used 500 ug, 1000ug and 2000ug total protein to start with. Since my protein has His6 tag I use monoclonal antibody for western detection. I see a lot of bands after IP on Western blotting without seeing the expected size band. What should I do? I dont know where am I going wrong.PLEASE HELP
Some ab don't work for IP and do work for WB, or the other way around. Since it works in both WB and IHC, two protocols which involve protein denaturation, it may be possible that the epitope that is recognized by the ab is actually hidden in the native form. If so, you could try "hardening" your IP conditions, such as adding more salt. But i'm really not sure this would work.