• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH    ₂ (Catalog # ). The specific activity is >30 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Human embryonic kidney cell, HEK293-derived Thr29-Thr461, with a C-terminal Fc tag

• Accession #

• N-terminal Sequence    
  Analysis

  Thr29 (pro-mature) and Tyr47 (mature)

• Predicted Molecular Mass

  75 kDa

• SDS-PAGE

  86-104 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 100 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0

• Recombinant Human Coagulation Factor IX (rhFactor IX) (Catalog # 9260-SE)

• Bacterial Thermolysin (Thermolysin) (Catalog # )

• Substrate: Fluorogenic Peptide Substrate II, MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)NH₂ (Catalog # )

• 1,10-Phenanthroline (Sigma, Catalog # 320056)

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rhFactor IX at 100 μg/mL wih 10 μg/mL Thermolysin in Assay Buffer.

2. Incubate at 37 °C for 90 minutes.

3. Stop the reaction by adding 1,10-Phenanthroline to the reaction tube at a final concentration of 10 mM.

4. Dilute activated rhFactor IX to 4 ng/μL in Assay Buffer.

5. Dilute Substrate to 20 μM in Assay Buffer.

6. Load 50 μL of the 4 ng/μL rhFactor IX into a black well plate, and start the reaction by adding 50 μL of 20 μM Substrate.  Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL 20 μM Substrate.

7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.

8. Calculate specific activity:

      *Adjusted for Substrate Blank
      **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975)

Per Well:

• rhFactor IX: 0.2 μg

• Substrate: 10 μM

Background: Coagulation Factor IX

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 (1). Factor IX consists of a Gla domain, two tandem EGF-like domains, an activation peptide, and an S1 serine protease domain (2). 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 Gla domain is modified by Vitamin K-dependent gamma-carboxylation and mediates the association of Factor IX with phospholipid bilayers (3, 4). The activation peptide is removed by Factor XIa mediated cleavage, resulting in heavy and light chains that remain disulfide-linked (5). Factor IX can also be activated by proteolytic factors in multiple snake venoms (6, 7). Active Factor IX associates with Factor VIIIa on the platelet surface where it cleaves and activates Factor X, leading to Fibrin deposition and clot formation (8-10). The human Factor IX gene is highly polymorphic, and Hemophilia B can be caused by X-linked deficiency of Factor IX activity (11-14).

• References:

1. Smith, S.A. et al. (2015) Crit. Rev. Biochem. Mol. Biol. 50:326.

2. Kurachi, K. and E.W. Davie (1982) Proc. Natl. Acad. Sci. USA 79:6461.

3. Rawala-Sheikh, R. et al. (1992) Blood 79:398.

4. Freedman, S.J. et al. (1996) J. Biol. Chem. 271:16227.

5. Di Scipio, R.G. et al. (1978) J. Clin. Invest. 61:1528.

6. Takeya, H. et al. (1992) J. Biol. Chem. 267:14109.

7. Sajevic, T. et al. (2011) Toxicon 57:627.

8. Ahmad, S.S. et al. (2003) J. Thromb. Haemost. 1:2348.

9. Mathur, A. et al. (1997) J. Biol. Chem. 272:23418.

10. van Dieijen, G. et al. (1985) Thromb. Haemost. 53:396.

11. Aguilar-Martinez, P. et al. (1994) Hum. Genet. 94:287.

12. Davis, L.M. et al. (1987) Blood 69:140.

13. Bertina, R.M. et al. (1990) J. Biol. Chem. 265:10876.

14. Girolami, A. et al. (2015) Semin. Thromb. Hemost. 41:359.

• Entrez Gene IDs:

  2158 (Human); 14071 (Mouse); 24946 (Rat)

• Alternate Names:

  Christmas Factor; Coagulation Factor IX; EC 3.4.21; EC 3.4.21.22; F9; factor 9; FIX; HEMB; MGC129641; MGC129642; P19; Plasma Thromboplastic Component; Plasma thromboplastin component; PTC; THPH8