Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter
1 Promega Corporation 2800 Woods Hollow Road, Madison, WI 53711, USA
2 Promega Biosciences 277 Granada Drive, San Luis Obispo, CA 93401, USA
BMC Cell Biology 2008, 9:17 doi:10.1186/1471-2121-9-17Published: 2 April 2008
Additional file 1:
Distinguishing cell surface and internalized HaloTag protein. Pre trypsin exposure, live HEK293 cells sequentially labeled with HaloTag 488 and TMR ligands show spatial separation of the green surface protein around the red internal protein, and some of the original green surface pool has internalized. After trypsin, the external HaloTag 488 ligand is stripped over 200 and 400 seconds and the internal HaloTag TMR ligand and internalized HaloTag 488 ligand are preserved. Cell images were generated on an Olympus FV500 confocal microscope in sequential mode using appropriate filter sets.
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Real-time translocation of proteins using HaloTag Technology. Timelapse imaging of live U2OS cells sequentially labeled with HaloTag 488 and TMR ligands shows real-time translocation of both protein pools. After labeling, time 0 shows spatial separation of the green surface protein around the red internal protein. Imaging every 20 minutes over 17 hours first shows the green surface pool internalizing and the red internal pool trafficking to the surface, then the original green surface pool inside and the red pool on the membrane, and finally the red surface pool internalizing.
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