PathHunter® Protease Protein Secretion Assay Platform

PathHunter products include novel, in vivo applications of Enzyme Fragment Complementation (EFC) technology to study protein secretion. A small 4kDa-complementing fragment of β-galactosidase (β-gal), termed ProLabel, is expressed as a fusion to a target protein of interest, which is secreted and released in the cell supernatant upon pathway activation. The secreted protein can be detected when the ProLabel complements with the larger enzyme acceptor (EA) fragment of β-gal (present in the secretion detection kit) to form functional β-gal enzyme. Enzyme activity is measured using a chemiluminescent substrate. Signal generation is directly proportional to the amount of cleaved substrate.

Technology Principle

PathHunter™ PCSK9 is a cell-based assay designed to  measure the secreted form of PCSK9. Signal is generated in the presence of a chemiluminescent substrate when the secreted PCSK9-PL and exogenous EA (enzyme acceptor) in the secretion detection kit complement to form active β-galactosidase enzyme (EFC).

Measure specific secretion of ProLabel-tagged proteases

Brefeldin A Inhibition Using PCSK9 Assay

EAstern Blot Analysis of PCSK9

Effect of non-specific secretion inhibitor Brefeldin A (BFA) on the PathHunter PCSK9 assay. This experiment demonstrates that BFA inhibits the secretion of PCSK9.
Measure PCSK9 secretion by EAstern Blot: In this experiment, complementary EA chemiluminescent enzyme substrate is used to detect the band containing the PL-tagged PCSK9. This experiment confirms that the EFC activity detected in the supernatant is solely due to the secreted form of PCSK9.

Features and Benefits:

  • Cell-based functional assay
  • Ideal for high-throughput screening
  • Chemiluminescent assay reduces false positives
  • Simple, two addition protocol