Active Coagulation Factor IX (F9)

Coagulation Factor IX, also known as Christmas Factor, is a secreted by the liver and plays a key role in the activation of the intrinsic clotting cascade. Factor IX consists of a Gla domain, two tandem EGF-like domains, an activation peptide, and an S1 serine protease domain. Mature human Factor IX shares approximately 81% amino acid sequence identity with mouse and rat Factor IX. Alternative splicing generates an additional isoform that lacks the first EGF-like domain. The activity of recombinant rabbit Coagulation Factor IX is measured by its ability to cleave a fluorogenic peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 in the assay buffer 100 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 8.0. The Coagulation Factor IX is diluted to 100 ug/ml in assay buffer, then activated with a final concentration of 10 ug/ml Thermolysin at 37 ℃ for 90min. Adding a final concentration of 10 mM 1,10 Phenanthroline to stop the activation. The activated Coagulation Factor IX is diluted to 6 ug/mL in assay buffer. Loading into a black well plate 50 µL of 6 ug/mL Coagulation Factor IX and start the reaction by adding 50 µL of 20 uM substrate, with a substrate blank containing 50 µL assay buffer, 50 µL substrate, and no Coagulation Factor IX. Then read at 320/405 nm in kinetic mode for 5 minutes. The specific activity of recombinant rabbit Coagulation Factor IX is > 20 pmol/min/µg.