- Protein homodimerization—the interaction of two identical protein subunits—is a fundamental mechanism in regulating protein function, stability, and signaling.
- A widely adopted method to study this interaction in vivo involves tagging the same protein with two different epitope tags (e.g., FLAG and HA) and assessing their co-association via Co-IP.
- The physical interaction is demonstrated by the ability to immunoprecipitate one tagged variant and detect the other in the immunoprecipitate.
Experimental Workflow:
- Construct Design and Expression:
- Clone the gene of interest into two separate mammalian expression vectors, fusing one with an epitope tag such as FLAG, and the other with a different tag (e.g., HA).
- Co-transfect the expression constructs into mammalian cells (e.g., HEK293T or HeLa).
- Protein Expression and Cell Lysis:
- After sufficient expression (typically 24–48 hours post-transfection), lyse the cells using a mild, non-denaturing lysis buffer (e.g., NP-40 or Triton X-100 based buffer) containing protease inhibitors.
- Clarify lysates by centrifugation to remove insoluble debris.
- Co-Immunoprecipitation (Co-IP):
- Incubate the cleared lysate with beads conjugated to an antibody specific for one epitope tag (e.g., anti-FLAG).
- Wash the beads extensively to remove non-specifically bound proteins.
- Detection via Western Blot:
- Elute immunoprecipitated proteins and separate by SDS-PAGE.
- Perform Western blotting using antibodies specific to both tags (e.g., anti-FLAG and anti-HA).
- Detection of both the immunoprecipitated protein and its tagged partner provides evidence of homodimer formation.
- Controls and Validation:
- Include single-transfection controls and empty vector controls to assess background binding.
- Optionally, include dimerization-deficient mutants to validate specificity.
Interpretation: Co-detection of both tagged proteins in the immunoprecipitate confirms physical association, supporting the presence of homodimers or higher-order oligomers under physiological conditions. The assay is amenable to quantitative and mutational analysis and provides a reliable in vivo method to assess self-association of proteins.
