Revealing protein topology and subcellular localization. (a) Diagram of separately labeled surface and internal protein pools of β1Int-HaloTag, and trypsin exposure to strip the surface labeled pool. (b) Pre trypsin treatment, live HEK293 cells sequentially labeled with HaloTag 488 and TMR ligands show spatial separation of protein pools depicted by a green rim around a red interior. After trypsin, the external HaloTag 488 ligand is stripped over 50 and 450 seconds and the internal HaloTag TMR ligand is preserved. (c) Diagram of β1Int-HaloTag and GFP co-expression in TMR-labeled cells, and digitonin exposure to permeabilize membrane. (d) HeLa cells transfected with β1Int-HaloTag and GFP were labeled with HaloTag TMR ligand. Co-expression of the 2 proteins, shown by yellow overlay (arrow), is present pre digitonin treatment, but after digitonin most GFP diffuses out of the cells over 360, 440 and 590 seconds and β1Int-HaloTag remains inside (arrow). Cell images were generated on an Olympus FV500 confocal microscope in sequential mode using appropriate filter sets.