详细说明
- Purity>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to be used as a protein substrate for ADAMTS13. >50% of rhvWF-A2 is cleaved as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
- SourceE. coli-derived Asp1498-Val1665, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass20 kDa
- SDS-PAGE18 kDa, reducing conditions
| 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.
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- Assay Buffer: 50 mM Tris, 2 mM CaCl2, 0.01% (w/v) Brij-35, pH 8.5
- Recombinant Human ADAMTS13 (rhADAMTS13) (Catalog # )
- 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 ( or ).
- rhADAMTS13: 10 µg/mL (0.5 µg)
- rhvWF-A2: 100 µg/mL (5 µg)
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.
- References:
- 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.
- Long Name:von Willebrand Factor A2 Domain
- Entrez Gene IDs:7450 (Human); 22371 (Mouse); 116669 (Rat)
- Alternate Names:F8VWF; VWD; VWF von Willebrand factor; VWF; vWFA2; vWF-A2
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