Recombinant Human Coagulation Factor XI Protein, CF
Recombinant Human Coagulation Factor XI Protein, CF Summary
Glu19-Val625, with a C-terminal 10-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl.|
|Reconstitution||Reconstitute at 100 μg/mL in sterile 50 mM Tris, 10 mM CaCl2 and 150 mM NaCl, pH 7.5.|
|Shipping||The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
- Assay Buffer: 50 mM Tris, 250 mM NaCl, 1 mM EDTA, pH 7.5
- Recombinant Human Coagulation Factor XI (rhFactor XI) (Catalog # 2460-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- EDTA, pH 8.0 (Sigma, Catalog # E-4884), 0.5 M stock in diH2O
- Substrate: t-Butyloxycarbonyl-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin (Bachem, Catalog # I-1100), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rhFactor XI at 100 µg/mL with 10 µg/mL Thermolysin in Activation Buffer.
- Incubate at 37 °C for 1 hour.
- Stop reaction with EDTA at a final concentration of 40 mM in Activation Buffer.
- Dilute incubated rhFactor XI to 4 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- In a plate, load 50 µL of 4 ng/µL rhFactor XI, and start the reaction by adding 50 µL of 200 µM Substrate.
- Include a Substrate blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
- rhFactor XI: 0.2 µg
- Thermolysin: 0.02 µg
- Substrate: 100 µM
Background: Coagulation Factor XI
Coagulation factors XI and XIa refer to the pro and active forms of the same protease, respectively (1). Factor XI is synthesized in the liver and circulates in the plasma as a disulfide bond-linked dimer complexed with high molecular weight kininogen. Factor XI is converted into XIa via either the contact phase of blood coagulation or thrombin-mediated activation on the platelet surface. The resulting XIa converts factor IX into IXa, which subsequently activates factor X into Xa. Factor Xa in turn activates factor II/thrombin to complete the coagulation cascade. Patients with factor XI deficiency are prone to excessive bleeding after hemostatic challenge. There are two alternative splicing forms. Isoform 1 corresponds to the circulating plasma factor XI and isoform 2 is produced by platelets and megakaryocytes but absent from other blood cells (2). The 625 amino acid precursor of isoform 1 consists of a signal peptide (residues 1 to 18) and the mature chain (residues 19 to 625). The mature chain (XI) can be further processed into the heavy chain (residues 19 to 387) and the light chain (residues 388 to 625) (XIa). The purified rhFactor XI corresponds to isoform 1 (residues 19 to 625), which can be activated by treatment with thermolysin under the conditions described in the Activity Assay Protocol.
- Wash, P.N. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1651.
- Hsu, T.C. et al. (1998) J. Biol. Chem. 273:13787.
Citation for Recombinant Human Coagulation Factor XI Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Molecular intercommunication between the complement and coagulation systems.
Authors: Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, Acker B, Bruckner UB, Nilsson B, Gebhard F, Lambris JD, Huber-Lang M
J. Immunol., 2010;185(9):5628-36.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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