Spatial and temporal separation of proteins using HaloTag Technology. (a) HEK293 cells stably expressing β1Int-HaloTag were sequentially labeled with the HaloTag 488 and TMR ligands and then rinsed and lysed. Lysate was non-glycanase treated (lane 2), O-glycanase treated (lane 3) or N-glycanase treated (lane 4) and analyzed by SDS-PAGE with a ladder (lane 1). (b) HEK293 cells stably expressing β1Int-HaloTag were labeled with the HaloTag TMR ligand alone (lane 1), the HaloTag 488 ligand alone (lane 2), or sequentially with the HaloTag 488 and TMR ligands (lanes 3–9). Cells were rinsed and lysed at 0 minutes (lanes 1–3) or 0.5, 1, 2, 4, 8 and 12 hours (lanes 4–9) after labeling, and lysate was run on SDS-PAGE. (c) HeLa cells expressing β1Int-HaloTag were sequentially labeled with the HaloTag 488 and HaloTag TMR ligands and imaged immediately after labeling to show spatially separated protein pools. (d) Cells were re-imaged 12 hours after labeling to show temporal translocation of both protein pools. Cell images were generated with Olympus FV500 confocal microscope in sequential mode using appropriate filter sets.