Recombinant Human FXI (carrier-free)

Product Details

Source

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.

Purity

>90%, as determined by Coomasie stained SDS-PAGE

Formulation

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

Concentration

Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)

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.

Activity

Thermolysin-activated human FXI hydrolyzes a fluorogenic substrate, Boc-IEGR-AMC, with a specific activity value greater than 200 pmol/µg/min.

Application

Bioassay

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

1. Activation Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35.
2. Assay Buffer: pH 8.0, 50 mM Tris, 0.25 M NaCl.
3. Activation Stop Solution: 500 mM EDTA solution at pH 8.0.
4. Bacterial Thermolysin (TLN).
5. Human FXI
6. FXI substrate:  Boc-IEGR-AMC

 

Assay Procedures

1. Prepare 20 µg/mL of TLN solution in the Activation Buffer.
2. 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.
3. Incubate at 37 °C for 1 hour.
4. Stop reaction with equal volume of Activation stop solution. At this moment, hFXI is at 50 µg/mL.
5. Dilute activated hFXI to 2 µg/mL (2 ng/µL) with Assay Buffer.
6. Dilute the substrate stock at 200 µM in Assay Buffer.
7. 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.
8. Include a Substrate blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
9. 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 tech@biolegend.com.

Antigen Details

Structure

Homodimer

Distribution

Hep G2 Cell line

Function

Intrinsic pathway of Blood coagulation, Hemostasis

Interaction

FIX, TFPI, antithrombin, C1-inhibitor

Ligand/Receptor

Heparin/Heparan Sulfate, FXIIa, Thrombin, FIX, Platelet glycoprotein GP1b

Bioactivity

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

UniProt

View information about FXI on UniProt.org

Documentation

• Technical data sheet
• Certificate of Analysis
• Material Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

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.

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.

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.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)

Certificate of Analysis