Overview
- Homodimerization implies that identical or nearly identical protein monomers associate under physiological conditions.
- By engineering two expression constructs encoding the same protein—each fused with a distinct epitope tag (e.g., FLAG and HA)—one can assess whether the two tagged versions interact in vivo. If the two proteins co-immunoprecipitate, it strongly supports the formation of homodimers or higher-order oligomers.
- This method provides a simple, robust, and direct approach to demonstrate homodimerization in a native-like mammalian context, especially when complemented by mutational analysis, reciprocal Co-IP, or FRET-based confirmation.
Objective: To determine whether a protein forms homodimers in mammalian cells by co-expressing two differently epitope-tagged versions of the same protein and testing for their physical association using co-immunoprecipitation (Co-IP).
Requirements
- Mammalian expression vectors: Protein X-FLAG, Protein X-HA
- Mammalian cells (e.g., HEK293T or HeLa)
- Transfection reagent (e.g., Lipofectamine 3000)
- Lysis buffer (e.g., NP-40 or Triton X-100 based; non-denaturing)
- Protease inhibitors
- Anti-FLAG antibody and FLAG-beads (e.g., anti-FLAG M2 magnetic beads)
- Anti-HA antibody (for Western blot)
- SDS-PAGE and Western blotting reagents
Procedure
Step 1: Construct Preparation
- Clone the gene of interest (e.g., Protein X) into two separate expression vectors. One construct should carry an N- or C-terminal FLAG tag; the other an HA tag.
- Confirm expression and tag placement do not interfere with protein folding or function.
Note: Epitope tags should not interfere with dimerization interfaces—terminal positioning should be chosen based on known structure/function.
Step 2: Transfection
- Co-transfect mammalian cells with both constructs (Protein X-FLAG + Protein X-HA).
- Include control groups: Single transfections (FLAG only or HA only) and Empty vector controls.
Step 3: Cell Lysis
- Harvest cells 24–48 hours post-transfection.
- Lyse cells in ice-cold non-denaturing lysis buffer (e.g., 50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; protease inhibitors).
- Incubate on ice and clear lysate by centrifugation.
Step 4: Co-Immunoprecipitation
- Incubate cleared lysate with anti-FLAG magnetic beads (or agarose beads) at 4°C for 2–4 hours.
- Wash beads thoroughly with lysis buffer to remove non-specific binders.
Step 5: Elution and Western Blot
- Elute bound proteins by boiling beads in SDS sample buffer.
- Separate proteins by SDS-PAGE.
- Perform Western blot:
- Probe with anti-FLAG antibody to confirm pulldown of FLAG-tagged protein.
- Probe with anti-HA antibody to detect co-immunoprecipitated HA-tagged protein.
Interpretation
- Detection of the HA-tagged version of Protein X in the FLAG pulldown strongly suggests homodimerization.
- Lack of HA signal in control lanes (e.g., FLAG-only transfection) confirms specificity.
- Further controls (e.g., using a dimerization-defective mutant) can validate the interaction mechanism.
