Promega Corporation

Tracking GPI-Anchored Proteins Using a Novel Multifunctional Protein Labeling...

Tracking GPI-Anchored Proteins Using a Novel Multifunctional Protein Labeling Technology Scientific Poster

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Abstract

Chad Zimprich1, Soshana Behrstock1, Mark McDougall2, Natasha Karassina1, Randy Learish1, Dieter Klaubert2, Keith V. Wood1, Georgyi V. Los1
1Promega Corporation, Madison, WI 53711,
2Promega Biosciences, Inc., San Luis Obispo, CA 93401

Here we demonstrate the HaloTag® Protein reporter fused to truncated human CD59 at the amino terminus can be efficiently expressed on the extracellular surface of mammalian cells. Using a non-permeable HaloTag® Alexa488 Ligand and a permeable HaloTag® TMR Ligand, we were able to separate the surface pool and the intracellular pool of the fusion protein. We were also able to monitor bidirectional trafficking of the HaloTag® GPI fusion chimera in real time and demonstrate the effect of different conditions on trafficking. Data of immunocytochemical analysis using anti-HaloTag® antibody confirmed our live cell imaging results. Fluorescently labeled protein pools were further analyzed using SDS-PAGE, fluoroimaging, and Western blot. As was expected, only the fusion proteins containing the N-terminal signal peptide and the proper GPI-anchoring sequence can be labeled with both cell-permeant and -impermeant ligands. Taken together our results indicate that the HaloTag® Technology can be used to study trafficking of GPI-anchored proteins and can be applied to both life science research and potentially drug development research.

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  • Part# PS046
  • Printed in USA.

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