I transfected COS-7 cells with my plasmids of interest and performed co-IP using mouse anti-GFP for IP and mouse anti-myc for IB. I got positive results, confirming that my proteins of interest interact. However, as expected, I also observed two extra bands in the co-IP samples corresponding to the heavy and light chains of the anti-mouse antibody, since both antibodies are from the same species.
To avoid these extra bands, I repeated the experiment using mouse anti-GFP for IP and rabbit anti-myc for IB. However, I observed nonspecific bands in all samples, including co-IP, input, and control (non-transfected), as shown in the image. Does anyone know why this is happening?
I tried blocking with non-fat skim milk and BSA, separately, but the issue persisted.
Thank you in advance!
Hello,
This is a common problem with small epitope-tags like anti-myc or anti-HA, as anti-myc or anti-HA antibodies can sometimes recognize proteins in the cell lysate that have similar epitopes to the myc or HA tags. For example, endogenous proteins in mammalian cells or yeast may contain sequences that mimic HA or Myc epitopes. I got similar problems while performing co-IP with anti-HA tag but these were specific to cell lysate. They could also be degraded products of the protein as these tags sometimes lead to proteasomal degradation. I used anti-HA antibody (monoclonal) to detect my protein and the problem was consistent, although anti-GFP did not yield these bands specific to the lysate. As you are receiving these bands in non-transfected negative controls, these are specific to your lysates. It is better to change the type of antibody or epitope tagging.
Best wishes
This may happen if you use BSA as your blocking agent. While increasing the sensitivity of the band(s) of interest, it may also increase the number of background bands, most likely due to reduced stringency conditions of the BSA. To reduce background bands (typical with polyclonal rabbit antibodies as they contain multiple idiotypes as opposed to a monoclonal antibody which has only a single idiotype) try using 5% non-fat milk. While this may reduce background bands, it could also reduce the band of interest, to the point where you may not detect it. I prefer to have too many bands than too few as I don't want to miss the target of interest, especially if the signal is inherently weak.
Hello, Khalood,
To avoid interference from heavy and light chains in an IP experiment, you can choose one of the following approaches:
Hope this helpful! Feel free to reach out anytime if you have any other questions or need more details.
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