Recombinant Human ADAM8 Protein, CF

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

Recombinant Human ADAM8 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH₂ (Catalog # ES003). The specific activity is >1 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human ADAM8 protein
  Met1-Pro497, with a C-terminal 6-His tag
• Accession #: P78325
• N-terminal Sequence: Glu158
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ADAM8
• 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: 38 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: 42 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 Glycerol, Tris, NaCl and CaCl₂.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • 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
  1. Dilute rhADAM8 to 400 μg/mL in Assay Buffer.
  2. 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.
  3. Incubate at 37 °C for 30 minutes.
  4. Stop reaction by adding Phosphoramidon to a final concentration of 0.05 mM.
  5. Incubate at room temperature for 15 minutes.
  6. Dilute activated rhADAM8 to 40 ng/μL in Assay Buffer.
  7. Dilute substrate to 40 μM in Assay Buffer.
  8. 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.
  9. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
  10. 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).

  Per Well:
  • rhADAM8: 2.0 μg
  • Substrate: 20 μM

Notes

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

Alternate Names for Recombinant Human ADAM8 Protein, CF

• a disintegrin and metalloproteinase domain 8
• ADAM 8
• ADAM metallopeptidase domain 8
• ADAM8
• CD156a antigen
• CD156a
• CD156human leukocyte differentiation antigen
• Cell surface antigen MS2
• EC 3.4.24
• EC 3.4.24.-
• MGC134985
• MS2
• MS2disintegrin and metalloproteinase domain-containing protein 8

Background

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.

1. Yoshiyama, K. et al. (1997) Genomics 41:56.
2. Moss, M.L. and J.W. Bartsch (2004) Biochemistry 43:7227.
3. Fourie, A.M. et al. (2003) J. Biol. Chem. 278:30469.
4. Naus, S. et al. (2004) J. Biol. Chem. 279:16083.
5. Choi, S.J. et al. (2001) J. Bone Miner. Res. 16:814.
6. King, N.E. et al. (2004) Am. J. Respir. Cell Mol. Biol. 31:257.

Publications for ADAM8 (1031-AD)(7)

Publications using 1031-AD

• Mineva, ND;Pianetti, S;Das, SG;Srinivasan, S;Billiald, NM;Sonenshein, GE; A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer Pharmaceutics 2024-04-13 [PMID: 38675197] (Bioassay, N/A)
• Pianetti, S;Miller, KD;Chen, HH;Althouse, S;Cao, S;Michael, SJ;Sonenshein, GE;Mineva, ND; ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients Cancer cell international 2023-08-11 [PMID: 37568162] (Bioassay, Human)
• Mi, Z;Kuo, MC;Kuo, PC; RNA Aptamer Targeting of Adam8 in Cancer Growth and Metastasis Cancers 2023-06-20 [PMID: 37370863] (Bioassay, N/A)
• MW Johansson, M Khanna, V Bortnov, DS Annis, CL Nguyen, DF Mosher IL-5-stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8-dependent migration Clin. Exp. Allergy, 2017-05-05;0(0):. 2017-05-05 [PMID: 28378503] (Bioassay, Human)
• 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 ADAM8 as a drug target in pancreatic cancer. Nat Commun, 2015-01-28;6(0):6175. 2015-01-28 [PMID: 25629724] (Bioassay, Human)
• McDonald A, Dibble J, Evans E, Millhauser G A new paradigm for enzymatic control of alpha-cleavage and beta-cleavage of the prion protein. J Biol Chem, 2013-11-18;289(2):803-13. 2013-11-18 [PMID: 24247244] (Enzyme Assay, Bacteria - E. Coli)
• 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 Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding. J. Immunol., 2007-06-15;178(12):8053-63. 2007-06-15 [PMID: 17548643] (Bioassay, Human)

Bioinformatics

• Gene Symbol: ADAM8
• Uniprot:
  • P78325()