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Abstract for Correlating Cellular Imaging of a p65-fusion
Reporter with Protein Interactions Using the HaloTag® Technology
Randall Learish1, Natasha Karassina1, Georgyi V. Los1, Chad
Zimprich1, Mark G. McDougall2, Dieter H. Klaubert2, Marjeta Uhr1,
Danette Hartzell1, Nidhi Nath1, Gediminas Vidugiris1, Robert F.
Bulleit1, Keith Wood1
1Promega Corporation, Madison, WI 53711
2Promega Biosciences Inc., San Luis Obispo, CA 93401
The study of complex protein networks in living cells is
facilitated by the use of technologies which
offer multiple applications. Here we study a cell-based model of the
NFκB signaling pathway using a reporter-fusion protein technology to
both functionally label, and immobilize and capture, p65 and its
binding partners. Fusion constructs were made using the HaloTag®
Interchangeable Labeling Technology and expressed by transient or
stable transfection in mammalian cell lines. p65 fusion
protein was immobilized onto HaloLink™ agarose beads without prior
purification steps. This approach provided for successful capture of
the p65 fusion protein as well as co-capture of its binding partner IκB, as demonstrated by western blotting. Fusion protein was also
captured using streptavidin-coated beads following labeling of the
p65-fusion with a biotinylated HaloTag® ligand. The p65-fusion was
visualized in the cytoplasm of living cells following transfection
and labeling with fluorescent ligands. Following fluorescent
labeling and treatment with TNFα in vivo, nuclear
translocation of the p65-fusion construct was visualized by time
lapse confocal imaging. At intervals after treatment, lysates were
obtained, fractionated into nuclear and cytoplasmic pools, and
probed. Western blot analysis revealed a time dependant degradation
and reappearance of IκB in the cytoplasm, as well as correlative
evidence for translocation of the p65-fusion protein into the
nucleus. Finally, the same technology was applied to study the DNA
promoter binding activity of the p65-fusion protein by cross-linking
TNFα treated samples and then capturing DNA fragments using
HaloLink™ resin. The covalent attachment and rapid binding rate of
the HaloTag® ligands to the reporter protein has made possible the
development of a large set of applications that span the areas of
imaging, high content analysis and proteomics. Here the versatility
of the technology permitted the generation of a broad set of data
using a single fusion construct to study NFκB signaling in vitro and
in vivo. |
