I have been trying for months to determine if proteins "X" and "Y" co-immunoprecipitate. However, whenever I run a negative control experiment that contains cell lysate and protein A magnetic beads (no antibody conjugated to the beads) and run my samples on a western, my negative controls always come up positive no matter whether I am probing for protein "X" or "Y." It have been troubleshooting the idea that the beads are non-specifically binding to my proteins, but is it possible that the tubes I am using are also binding non-specifically to my proteins? I do frequent washings throughout my Co-IP, but I never change the tube that contains my magnetic beads. Any thoughts?
I don't think it is the tubes. Overexpressed proteins show a tendency to non specifically bind to beads also. You can try blocking beads with BSA or something inert or may be use less of lysate (usually I do 30ul prA beads non magnetic for 500ug lysate). Washing at least 3times after the IP is important and may be you can try eluting protein complexes with low pH (0.1M glycine pH3.5 and neutralize with Tris-Cl pH8 so that you avoid taking the beads, and just boil the eluate in SDS loading buffer. Hope it helps.Goodluck
I agree with Bharat - it's not your tubes, it's your beads that are a culprit. As he suggested, try to block them better (BSA is a fine choice), use BSA throughout your procedures and elute with low pH glycine. Couple more suggestions: first, try changing the beads to sepharose. In my experience, they have lower non-specific binding capacity than the magnetic ones. Spinning vs magnet in that case is not much of a sacrifice in convenience. And try to wash with buffers containing higher concentrations of detergents (and harsher ones - like Triton instead of Tween, etc). Good luck indeed - you might need it!
I have a couple of questions:
1. Are you pre-washing your beads with PBS or as recommended by manufacturer?
2. Are you allowing binding in the lysis buffer (what kind are you using?)
This is important because these usually have detergents and will diminish
unspecific binding.
3. Are you using continuous rotation during binding (in my experience this is very
important to obtain perfect binding of your proteins and avoid unspecific binding)
4. Are you washing with constant rotation? (it doesn't work if you don't)
I second Vadims suggestion to use different beads. The magnetics beads are very convenient, but i recently had the same problem you have and went back to agarose beads and spinning. This resolved my non-specific binding issue. Before trying other beads I had tried extensive blocking, harsher buffers, longer washes, and nothing seemed to solve my problems.
I would like to add that wash your beads at the end with high quality water twice. It may reduce the background significantly (depending upon the net charge on the protein(s) of interest).
Protein can no specifically bind to beads for sure. I sometimes increase the salt concentration to 200-250 mM NaCl to drive the protein onto the antibody. Try adding 0.005%tween20 .
Hey Charles, Did you get this problem solved? Even I am facing similar problem by with Protein A/G agarose beads and Sepharose beads (we tried both the beads from Santa cruz and Thermoscientific). I have tried to change all things possible and we are planning to try with a different buffer now. Please let me know if you have resolved your problem with COIP?
It would be of great help, thank you
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