Recombinant Human ADAM8 Protein, CF Summary
Met1-Pro497, with a C-terminal 6-His tag
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Glycerol, Tris, NaCl and CaCl2.|
|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 Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
- Recombinant Human ADAM8 (rhADAM8) (Catalog # 1031-AD)
- Bacterial Thermolysin (Catalog # 3097-ZN)
- Phosphoramidon (Catalog # EI006), 20 mM in methanol
- Fluorogenic Peptide Substrate III: MCA-Pro-Leu-Ala-Gln-Ala-Val-DPA-Arg-Ser-Ser-Ser-Arg-NH2 (Catalog # ES003)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhADAM8 to 400 μg/mL in Assay Buffer.
- Combine equal volumes 1.5 μg/mL thermolysin and diluted rhADAM8 so that the final concentration of rhADAM8 is 200 μg/mL and thermolysin is 0.75 μg/mL.
- Incubate at 37 °C for 30 minutes.
- Stop reaction by adding Phosphoramidon to a final concentration of 0.05 mM.
- Incubate at room temperature for 15 minutes.
- Dilute activated rhADAM8 to 40 ng/μL in Assay Buffer.
- Dilute substrate to 40 μM in Assay Buffer.
- Load 50 μL of 40 ng/μL rhADAM8 in a black well plate and start the reaction by adding 50 μL of 40 μM substrate. Include a control containing 50 μL Assay Buffer and 50 μL of 40 μM substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhADAM8: 2.0 μg
- Substrate: 20 μM
ADAM8, also known as cell surface antigen MS2 or CD156a, is a member of the ADAM family that contains a disintegrin and metalloprotease-like domain (1, 2). ADAM8 can cleave a variety of substrates and has been shown as a sheddase for the low affinity IgE receptor CD23 and the neural recognition molecule CHL1 (3, 4). Expression and regulation studies suggest that ADAM8 is a novel osteoclast stimulating factor and may play a role in asthma (5, 6). The 824 amino acid precursor of human ADAM8 consists of a signal peptide (residues 1 to 16), a pro peptide (residues 17 to 199), a metaloprotease domain (residues 200 to 400), a disintegrin-like domain (residues 408 to 494), a cysteine-rich region (residues 497 to 613), an EGF-like domain (residues 614 to 640), a transmembrane region (residues 656 to 676) and a cytoplasmic domain (residues 677 to 824). The purified rhADAM8 (residues 158 to 497) contains a part of the pro peptide and the entire metalloprotease and disintegrin-like domains. It can be activated and assayed under the conditions described in the Activity Assay Protocol.
- Yoshiyama, K. et al. (1997) Genomics 41:56.
- Moss, M.L. and J.W. Bartsch (2004) Biochemistry 43:7227.
- Fourie, A.M. et al. (2003) J. Biol. Chem. 278:30469.
- Naus, S. et al. (2004) J. Biol. Chem. 279:16083.
- Choi, S.J. et al. (2001) J. Bone Miner. Res. 16:814.
- King, N.E. et al. (2004) Am. J. Respir. Cell Mol. Biol. 31:257.
Citations for Recombinant Human ADAM8 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 4
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IL-5-stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8-dependent migration
Authors: MW Johansson, M Khanna, V Bortnov, DS Annis, CL Nguyen, DF Mosher
Clin. Exp. Allergy, 2017-05-05;0(0):.
Sample Types: Whole Cells
ADAM8 as a drug target in pancreatic cancer.
Authors: Schlomann U, Koller G, Conrad C, Ferdous T, Golfi P, Garcia A, Hofling S, Parsons M, Costa P, Soper R, Bossard M, Hagemann T, Roshani R, Sewald N, Ketchem R, Moss M, Rasmussen F, Miller M, Lauffenburger D, Tuveson D, Nimsky C, Bartsch J
Nat Commun, 2015-01-28;6(0):6175.
Sample Types: Whole Cells
A new paradigm for enzymatic control of alpha-cleavage and beta-cleavage of the prion protein.
Authors: McDonald A, Dibble J, Evans E, Millhauser G
J Biol Chem, 2013-11-18;289(2):803-13.
Species: Bacteria - E. Coli
Sample Types: Cell Lysates
Applications: Enzyme Assay
Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding.
Authors: Gomez-Gaviro M, Dominguez-Luis M, Canchado J, Calafat J, Janssen H, Lara-Pezzi E, Fourie A, Tugores A, Valenzuela-Fernandez A, Mollinedo F, Sanchez-Madrid F, Diaz-Gonzalez F
J. Immunol., 2007-06-15;178(12):8053-63.
Sample Types: Whole Cells
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Fluorogenic Peptide Substrates
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