Recombinant Human ADAMTS15 Protein, CF Summary
Product Specifications
Gly18-Cys682 (pro) & Phe213-Cys682 (mature), both with a C-terminal 10-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.
5149-AD
| Formulation | Supplied as a 0.2 μm filtered solution in MES and NaCl. |
| 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
- Dilution Buffer: 50 mM Tris, 0.02% (w/v) Brij-35, pH 7.5
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Human Aggrecan G1-IGD-G2 Domains (rhAggrecan) (Catalog # 1220-PG)
- Recombinant Human ADAMTS15 (rhADAMTS15) (Catalog # 5149-AD)
- SDS-PAGE and/or Western blot
- Dilute rhADAMTS15 to 25 μg/mL in Dilution Buffer.
- Incubate on ice for 4 hours.
- Dilute rhADAMTS15 to 20 µg/mL in Assay Buffer.
- Dilute rhAggrecan to 250 µg/mL in Assay Buffer.
- Combine equal amounts diluted rhAggrecan and diluted rhADAMTS15. As controls, combine equal amounts Aggrecan and Assay Buffer as well as equal amounts rhADAMTS15 and Assay Buffer.
- Incubate the reaction mixture and controls overnight at 37 °C.
- Combine 30 µL of reactions (and controls) with 26.2 µL reducing SDS-PAGE sample buffer.
- Analyze the cleavage by SDS-PAGE (load 15 µL per lane) followed by protein staining and/or Western blot.
- rhADAMTS15: 0.080 µg
- rhAggrecan: 1 µg
Reconstitution Calculator
Background: ADAMTS15
ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is a group of nineteen secreted zinc binding metalloproteases that is involved in development, inflammation, angiogenesis and several pathological conditions (1, 2). They are synthesized as zymogens which have a pro-domain that is removed by furin-like protein convertases. The mature secreted enzymes consist of catalytic, disintegrin-like, type I thrombospondin (TSP) motif, cysteine-rich, and spacer domains followed by zero to fourteen additional TSP motifs. Among ADAMTS proteases, ADAMTS1, -4, -5, -8, and -15 form a subfamily that possess aggrecanase activity (1-3). While ADAMTS4 and -5 (also known as aggrecanse-1 and -2, respectively) (4) have been well studied, the enzymatic activity of ADAMTS15 remains to be explored. It is suggested that ADAMTS15 may be involved in aggrecan degradation and breast cancer (5, 6). ADAMTS15 may also exhibit tumor suppressor activities since it contains genetic mutations in human colorectal carcinomas and restrains tumor growth and invasion in both in vitro and in vivo studies (7). The purified rhADAMTS15 is truncated and ends before the spacer domain.
- Porter, S. et al. (2005) Biochem. J. 386:15.
- Jones, G.C. and G.P. Riley (2005) Arthritis Res. Ther. 7:160.
- Nicholson, A.C. et al. (2005) BMC Evol. Biol. 5:11.
- Nagase, H. and M. Kashiwagi (2003) Arthritis Res. Ther. 5:94.
- Porter, S. et al. (2006) Intl. J. Cancer 118:1241.
- Kevorkian, L. et al. (2004) Arthritis Rheum. 50:131.
- Viloria, C.G. et al. (2009) Cancer Res. 69:4926.
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I used this protein as a standard for developing an ELISA in combination with pAb (cat. AF5149) and mAb (cat. MAB 5149). This combination works well for a sandwich ELISA detection human ADAMTS 14 in serum.
