Recombinant Human vWF-A2 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human vWF-A2 Protein, CF Summary

• Details of Functionality: Measured by its ability to be used as a protein substrate for ADAMTS13. >50% of rhvWF-A2 is cleaved as measured under the described conditions.
• Source: E. coli-derived human vWF-A2 protein
  Asp1498-Val1665, with an N-terminal Met and 6-His tag
• Accession #: NP_000543
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: VWF
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 20 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 18 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 2 mM CaCl₂, 0.01% (w/v) Brij-35, pH 8.5
  • Recombinant Human ADAMTS13 (rhADAMTS13) (Catalog # 4245-AD)
  • Recombinant Human vWF‑A2 (rhvWF‑A2) (Catalog # 2764-WF)
  • SDS-PAGE/Silverstain Reagents or equivalent
  1. Dilute rhvWF-A2 to 200 µg/mL in Assay Buffer.
  2. Dilute rhADAMTS13 to 20 µg/mL in Assay Buffer.
  3. Mix 25 µL of 200 µg/mL rhvWF-A2 with 25 µL of diluted rhADAMTS13. Prepare two Blanks by mixing 25 µL of 200 µg/mL rhvWF-A2 with 25 µL of Assay Buffer.
  4. Incubate the mixture for 2 hours at 37 °C. Incubate one blank at 37 °C for 2 hours while keeping the other Blank at -20 °C.
  5. Stop the reaction by adding 15 µL of the reaction mixture to 15 µL of SDS-PAGE sample buffer. Also combine 15 µL of the blanks with 15 µL SDS-PAGE sample buffer.
  6. Analyze the cleavage by SDS PAGE (Load 20 µL per lane) followed by silver staining (1 µg rhvWF-A2 per lane).
  7. The cleavage products can also be analyzed by Western blot, loading 0.1 µg/lane of rhvWF-A2 and using one of R&D Systems antibodies (MAB27641 or MAB2764) .
  Per Reaction:
  • rhADAMTS13: 10 µg/mL (0.5 µg)
  • rhvWF-A2: 100 µg/mL (5 µg)

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human vWF-A2 Protein, CF

• F8VWF
• VWD
• VWF von Willebrand factor
• vWFA2
• vWF-A2

Background

von Willebrand Factor (vWF) is a large, multimeric glycoprotein made by endothelial cells and megakaryocytes. The pre-pro-vWF protein contains 2813 amino acids (aa), which consists of 22 aa signal peptide, 741 aa propeptide and mature vWF monomer of 2050 aa (1‑4). The pro-vWF undergoes dimerization in the endoplasmic reticulum (ER) through C-terminal “cysteine-knot” (CK) domain. The pro-vWF dimmers are transported to Golgi and form multimers by forming disulfide bond in amino‑terminal region of the mature form. The proteolytic processing of pro-region also occurs in Golgi. The matured vWF is stored in Weibel-Pallade bodies in endothelial cells and granules in megakaryocytes and platelets. The unusually-large vWF (ulvWF) multimers released from cells are very efficient in binding to platelets to form thrombus. The population of these highly active ulvWF multimers is controlled by a specific protease, ADAMTS13, which cleaves between residues Tyr1605 and Met1606 in the A2 domain of vWF. In the plasma, vWF appears as a series of large and intermediate multimers with molecular masses from several thousand to 500 kDa. vWF also performs hemostatic functions (3‑5). In a high shear-stressed environment, vWF undergoes conformational change to expose a binding site for glycoprotein Ib alpha . As a result, vWF facilitates aggregation of platelets. In addition to platelet binding, vWF binds coagulation factor VIII to increase the lifetime of FVIII in plasma. The purified recombinant human vWF-A2 contains the A2 domain of vWF.

1. Sadler, J. E. (1998) Annu. Rev. Biochem. 67:395.
2. Ruggeri, Z. M. (2003) Cur. Opin. Hemat. 10:142.
3. Michiels, J. J. et al. (2006) Clin. Appl. Thromb. Hemost. 12:397.
4. Groot, E. et al. (2007) Cur. Opin. Hemat. 14:284.
5. Lenting, P. J. et al. (2007) J. Thromb. Haemos. 5:1353.

Bioinformatics

• Gene Symbol: VWF
• Uniprot:
  • NP_000543(Sheep)