Non-specific binding to immunoprecipitation beads - what to do to minimize it? (Oct/15/2008 )
I have two proteins (say, X and Z), which I believe interact. I have tagged them FLAG-X and HA-Z and incubated co-transfected lysates with anti-FLAG beads. When I WB for HA, I see it being pulled down.
The problem is that control also shows the same: HA-Z alone (without co-transfection with FLAG-X) can be pulled down with the FLAG beads.
Why is it so? What can I change in my protocol to minimize this non-specific binding? I use 0.5% Triton in the incubation buffer and wash the beads 4 times after incubation. This is my first Co-IP, so ANY suggestions would be highly appreciated.
You may try to increase the stringency (is that a word?! it is in french!) of your wash buffer. Try adding NaCl to different concentrations, until you can no longer detect the HA-Z protein but can still IP the FLAG-Y protein with the beads alone.
Hope this helps!
I have the same problem. Is your HA-Z protein size similar to those Ig bands? If so, can you still play with the lysis buffer stringency to solve this problem?