Why Do Two Bands Appear in SDS-PAGE After Co-IP to Detect Protein-Protein Interaction
Co-immunoprecipitation (Co-IP) is a widely used method for detecting protein-protein interactions. In brief, when researchers hypothesize that two proteins may interact, they can use an antibody specific to one of the proteins to immunoprecipitate it along with any proteins bound to it. The precipitated protein complex is then subjected to SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis), followed by analysis through Western blotting.
The appearance of two bands on the SDS-PAGE gel typically indicates the following:
1. “Bait” Protein
This is the protein targeted by the antibody during immunoprecipitation. It is usually a well-characterized protein of interest, selected to identify its potential interaction partners.
2. “Prey” Protein
If the “Bait” protein interacts with another protein, the interacting partner is also co-precipitated. This additional band represents the “Prey” protein that binds to the “Bait” protein.
To confirm that these two bands correspond to the proteins of interest, Western blot analysis is typically performed using antibodies specific to each protein. Additionally, appropriate experimental controls—such as no-antibody controls or irrelevant antibody controls—should be included to verify the specificity of the observed bands.
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