- Other Names
- Factor XI, FXI, Plasma Thromboplastin Antecedent, PTA, F11, Coagulation factor XI
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Human FXI is a plasma serine protease that is involved in contact activation pathway (intrinsic pathway) of blood congaguation by activation FIX. It is expressed as two alternative splicing forms. Isoform 1 is primarily synthesized by hepatocytes and circulates in a zymogen form. Isoform 2 is produced by platelets and megakaryocytes. Factor XI is converted into XIa via FXIIa, thrombin, and may undergo auto-activation in a presence of polyanions, e.g., inorganic polyphosphate polymers. It is a 160 kDa disulfide-linked homodimer in which each monomer contains a light chain (C-terminal domain) and heavy chain (N-terminal domain). The former is a serine-protease-like catalytic domain whereas the latter has four 90– or 91–amino acid repeats known as apple domains. The apple domains contain the FXI/FXIa binding sites for other molecules including thrombin, high molecular weight kininogen (HK), FIX, platelet glycoprotein GPIb, heparin/heparan sulfate, and FXIIa. The apple domain also contains the interface between the 2 monomers. The FXIa is physiologically inhibited mainly by plasma serpins including antithrombin and C1-inhibitor among others. However, it can neutralize tissue factor pathway inhibitor (TFPI) by proteolysis. FXI deficiency does not usually lead to spontaneous bleeding, but it is associated with an increased risk of bleeding when the haemostatic system is challenged. Epidemiological and animal model studies have associated FXI levels with the risk of thrombotic disease or septic survival advantage.Product Details
- Human FXI, amino acids Glu19-Val625 (Accession #NM_000128) with a C-terminal TG-8H-GGQ tag was expressed in CHO cells.
- Molecular Mass
- The 620 amino acid recombinant protein has a predicted molecular mass of approximately 69.5 kDa. The non-reduced and DTT-reduced proteins migrate at 160 and 70 - 80 kDa, repectively by SDS-PAGE.
- >90%, as determined by Coomasie stained SDS-PAGE
- 0.22 µm filtered protein solution is in pH 7.5 buffer containing 20 mM TRIS and 300 mM NaCl.
- Endotoxin Level
- Less than 1.0 EU per µg of protein as determined by the LAL method
- Lot-specific (please contact technical support for concentration and total µg amount, or use our Lookup tool if you have a lot number.)
- Storage & Handling
- Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.
- Thermolysin-activated human FXI hydrolyzes a fluorogenic substrate, Boc-IEGR-AMC, with a specific activity value greater than 200 pmol/µg/min.
- Application Notes
Human FXI Assay Procedures
Human FXI (hFXI) activity is measured by its ability to cleave a fluorogenic peptide substrate Boc-IEGR-AMC after hFXI is activated by thermolysin. The progress of the substrate cleavage is monitored by increase in intensity of fluorescence at 460 nm with excitation at 380 nm. This protein is in the latent form and needs to be activated for bioassay.
Materials and Buffers
- Activation Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35.
- Assay Buffer: pH 8.0, 50 mM Tris, 0.25 M NaCl.
- Activation Stop Solution: 500 mM EDTA solution at pH 8.0.
- Bacterial Thermolysin (TLN).
- Human FXI
- FXI substrate: Boc-IEGR-AMC
- Prepare 20 µg/mL of TLN solution in the Activation Buffer.
- Activate hFXI at 100 ug/mL with 10 µg/mL of TLN in activation buffer by adding equal volume of 20 µg/mL TLN solution into 200 ug/mL hFXI solution.
- Incubate at 37 °C for 1 hour.
- Stop reaction with equal volume of Activation stop solution. At this moment, hFXI is at 50 µg/mL.
- Dilute activated hFXI to 2 µg/mL (2 ng/µL) with Assay Buffer.
- Dilute the substrate stock at 200 µM in Assay Buffer.
- Load 50 µL of the activated hFXI in 2 µg/mL into a plate, and start a reaction by adding 50 µL of 2 mM of the substrate solution into each well.
- Include a Substrate blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
- Read the reaction progress by monitoring fluorescence at excitation and emission wavelength of 380 nm and 460 nm, respectively in kinetic mode for 5 minutes.
- The final human hFXI concentration is at 1 µg/mL (100 ng).
- The final substrate concentration at 100 µM.
- The final TLN concentration is at 0.1 µg/mL (10 ng), and EDTA at 5 mM.
BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at email@example.com.
Hep G2 Cell line
- Intrinsic pathway of Blood coagulation, Hemostasis
- FIX, TFPI, antithrombin, C1-inhibitor
- Heparin/Heparan Sulfate, FXIIa, Thrombin, FIX, Platelet glycoprotein GP1b
- Activate FIX to FIXa; Neutralize TFPI by proteolysis
- Molecular Family
- Enzymes and Regulators
- Antigen References
1. Weitz J. I. and Fredenburgh J. C. 2017. Front. Med. 4:19.
2. AI-Horani, R. A. and Desai, U. R. 2016 Expert Opin Ther Pat 26:323.
3. Puy C. et al. 2015. Blood 125:1488.
4. Geng Y. et al. 2013. Blood 121:3962.
5. Schumacher W. A. et al. 2010. Arterioscler Thromb Vasc Biol 30:388.
- Gene ID
- 2160 View all products for this Gene ID
- View information about FXI on UniProt.org
- Does specific activity of a recombinant protein vary between lots?
Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.
- Have your recombinants been tested for stability?
Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.
- How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
- Use formula Specific activity (Units/mg) = 10e6/ ED50 (ng/mL)
- How does the activity of your recombinant proteins compare to competitors?
We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!
- What is the specific activity or ED50 of my recombinant protein?
The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.