Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis |
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Authors: | Thorsten Seidel Daniel Schnitzer Dortje Golldack Markus Sauer Karl-Josef Dietz |
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Institution: | 1. Promega Corporation, 2800 Woods Hollow Road, Madison, WI, 53711, USA 2. Promega Biosciences, 277 Granada Drive, San Luis Obispo, CA, 93401, USA
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Abstract: | Background The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag®, to the extracellular domain of a truncated integrin. Results Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture. Conclusion The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells. |
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