Recombinant Human vWF-A2 Protein, CF Summary
Product Specifications
Asp1498-Val1665, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
2764-WF
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
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
- Dilute rhvWF-A2 to 200 µg/mL in Assay Buffer.
- Dilute rhADAMTS13 to 20 µg/mL in Assay Buffer.
- 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.
- 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.
- 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.
- Analyze the cleavage by SDS PAGE (Load 20 µL per lane) followed by silver staining (1 µg rhvWF-A2 per lane).
- 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).
- rhADAMTS13: 10 µg/mL (0.5 µg)
- rhvWF-A2: 100 µg/mL (5 µg)
Reconstitution Calculator
Background: vWF-A2
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.
- Sadler, J. E. (1998) Annu. Rev. Biochem. 67:395.
- Ruggeri, Z. M. (2003) Cur. Opin. Hemat. 10:142.
- Michiels, J. J. et al. (2006) Clin. Appl. Thromb. Hemost. 12:397.
- Groot, E. et al. (2007) Cur. Opin. Hemat. 14:284.
- Lenting, P. J. et al. (2007) J. Thromb. Haemos. 5:1353.
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