PathHunter™: High-throughput Assays for Protein Trafficking and Interactions Wins Technology Innovation Award

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Overview & Description
PathHunter™ products comprise a family of simple, robust cell-based assays and related tools designed to directly measure protein translocation, secretion, degradation, expression, and cell proliferation-without imaging or antibodies. These products may address cell-signaling cascades in which a target protein undergoes nuclear translocation, degradation, or secretion. A variety of targets can be measured, including transcription factors, nuclear hormone receptors, and kinases. Other unique PathHunter assays enable measuring mitotic activity in mammalian cells.

In PathHunter assays, a target protein fused to a ß-galactosidase (ß-gal) peptide (ProLabel™ tag) is expressed in the cytoplasm. Another portion of ß-gal (Enzyme Activator, or EA) is expressed only in the nucleus. When the two interact, active enzyme is formed generating signal. This method can detect protein translocation to the nucleus or nuclear envelope degradation during mitosis.

Key Features
• Homogeneous, chemiluminescent pathway profiling assays
• Precise, robust assays with high S:B
• No imaging, special equipment, antibodies, or GFP required
• No license fee

Homogeneous Antibody-free Assays
PathHunter products feature a novel, non-imaging approach based upon detecting complementation of two ß-galactosidase fragments, which generates a functional enzyme and a chemiluminescent signal. Consequently, no antibody labeling or imaging is required, and the assays are compatible with standard microplate readers. The basic PathHunter procedure is elegantly simple, and its homogeneous, two-step format offers high-sensitivity chemiluminescent detection.

Ready-to-use Assays or Custom Targets
For maximal convenience, PathHunter products include ready-to-use Biosensor Cell Lines. For optimal flexibility, engineered EA-Nuc Cell Lines and ProLabel™ Vectors can be combined to develop assays for a particular target protein known to undergo nuclear translocation.

PathHunter Biosensor Cell Lines are double-stable cell lines with the EA ß-gal fragment restricted to the nucleus, plus one of a variety of targets expressed as a ProLabel-tagged fusion protein in the cytoplasm.

PathHunter Assay Development Tools: EA-Nuc Cell Lines, Vectors and Detection Reagents may be combined to assay virtually any other target known to undergo nuclear translocation.

Assay Development

PathHunter is an open system. You can choose one of our ready to use Biosensor cell lines, or you can develop your own assay using a ProLabel-tagged target of interest and transfecting an EA-Nuc cell line. We also offer a series of expression vectors that express targets fused to ProLabel. This list is always expanding, please contact DiscoveRx for more information.