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Large bands (207kDa) showing up in CoIP western - (Jun/06/2008 )

I have had some issues with a CoIP for an AhR (about 95kDa) protein. I have cross-linked the pull-down ab (anti-AhR from abcam) to the G protein coated beads, added whole cell lystae (used RIPA, passed lysate through 21ga needle) and rotated lyasate with teh beads at 4C overnight. After the rotation, I wash the beads with TE (Tris-HCL with 10mM EDTA) 2 times, add loading dye and boil for 3-5 min at 95C. I then run a western and probe for my proteins……The problem is I have these bands greater than 207kDa. Could this be DNA that is messing up the CoIP????? Has anyone had this issue??


I have seen this in co-IPs very often.
It is the secondary antibody cross-reacting with IgGs (possibly other proteins)
used to IP that do not enter the gel (again may be other proteins).
To clear up this problem I do a control (I know, people HATE controls). I always do a Western on my IPed material
with the secondary antibody alone. Using the secondary alone you WILL see these bands.
If your primary IP antibody is a different species from your Western antibody this problem will be greatly diminished.
But seriously, do the control. I am 100% sure that this is the problem I have encountered exactly the same thing and fixed it by simply changing the species of the primary IP and Western. It is a non-specific interaction that you will see when you do the control for with your secondary alone for the Western.
PS. Do the control smile.gif