I conducted a co-immunoprecipitation using protein X antibody to determine if protein X is attached to protein Y. However, the results of my experiments were unusual. To ensure that the procedure was performed correctly, I conducted a control co-immunoprecipitation using protein Y antibody and another antibody that is not specific, which we'll refer to as Z. For the co-immunoprecipitation, I used Dynabeads™ Protein G and confirmed the proteins using WB.
The co-immunoprecipitation experiment using X antibody to pull out Y showed positive results for both X and Y. However, when the experiment was conducted using Y antibody to pull out X, it showed positive results only for Y and no X at all. Additionally, the immunoprecipitation of X and Y using Z antibody (which is nonspecific) showed positive results for both X and Y but the concentration was very weak. This suggests that there might be some issues with the experimental procedure. Can you suggest any measures to confirm the results with proper controls?
Hi Shimaa, it is not uncommon for co-IPs to only work in one direction. There can be a number of reasons such as the antibody and the interacting protein competing for the same binding site or different sensitivities of the respective antibodies. If it's possible, you may want to try a different antibody to your Y protein or move the tag if you are using one. Additionally, another good control would be to use your X antibody to conduct an IP in the absence of X protein expression to make sure that your pull-down of Y is dependent on its interaction with X (this only works if X is overexpressed or can be silenced).
Regarding your binding when you use a non-specific antibody, a lot of proteins will exhibit some degree of non-specific in IPs. If the levels are extremely low relative to the levels in the specific IP, this can usually be ignored. If it's a problem, you can try optimizing your wash step or pre-blocking the beads in BSA to see if you can reduce the non-specific binding.
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