3 extra bands show up on my western after an IP - (May/08/2013 )
Hi, I have done an IP with a mouse antibody to pull down my protein of interest. When i resolve this by SDS-PAGE and immunoblotting against the protein of interest (using the same antibody as the IP) I get 3 extra bands. I know that there should be 2 bands - the heavy and light chains of the IgG, but I get two smaller ones instead of one small light chain band. My IP protocol span the beads down and did clearing spins, but i didnt ever remove the beads, just suspended the pellet in SDS loading buffer and heat at 95C for 5 minutes. Can anybody shed some light on what the two smaller 'light chain' like bands are? I used A/G protein agarose beads.
you need to wash your beads more in the last steps before adding sds. It should be ok to get extra bands if they don't hide your protein of interest. Even IP can give extra bands.
I have also had protein G elute off beads (approx 25 kDa), but the proteinA/G you are using should run at about 50 IIRC.