Figure 1: Diagram of the Mediator-HaloTag pulldown process. A genetic construct encoding the HaloTag (HT) protein fused N-terminally to a Mediator subunit (MED) was transiently expressed in HEK293T cells, where it incorporates into Mediator. After cellular lysis, MED-HaloTag complexes were covalently captured on HaloLink resin. Gentle washes were performed to remove non-specific interactions while maintaining complexes. Interacting protein partners were eluted with 1% SDS and analysed either by mass spectrometry or Western blot. As a control, HaloTag alone protein was expressed and processed through the entire pull-down protocol identically to the MED-HT sample.