Recombinant Mouse ADAM10 Protein, CF

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

Recombinant Mouse ADAM10 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 >5 pmol/min/µg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived mouse ADAM10 protein
  Thr215-Glu673, with a C-terminal 10-His tag
• Accession #: O35598
• N-terminal Sequence: Thr215
• Structure / Form: Recombinant Mouse ADAM10 is prone to proteolytic cleavage at C-terminus. The predominant form of the purified protein lacks the His tag.
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Adam10
• 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: 52 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: 60 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in MES, NaCl, CaCl₂, ZnCl₂, and Brij-35.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile 25 mM Tris, pH 7.5.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 2.5 µM ZnCl₂, 0.005% (w/v) Brij-35, pH 9.0
  • Recombinant Mouse ADAM10 (rmADAM10) (Catalog # 946-AD)
  • Substrate: 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 rmADAM10 to 2 ng/µL in Assay Buffer.
  2. Dilute Substrate to 20 µM in Assay Buffer.
  3. In a plate load 50 µL of 2 ng/µL rmADAM10, and start the reaction by adding 50 µL of 20 µM Substrate to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
  4. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 30 minutes at 37 °C.
  5. Calculate specific activity using data from 15-30 minutes:

  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:
  • rmADAM10: 0.1 µg
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Mouse ADAM10 Protein, CF

• a disintegrin and metalloprotease domain 10
• a disintegrin and metalloproteinase domain 10
• AD10
• ADAM 10
• ADAM metallopeptidase domain 10
• ADAM10
• CD156c antigen
• CD156c
• CDw156
• disintegrin and metalloproteinase domain-containing protein 10
• EC 3.4.24
• HsT18717
• kuz
• Kuzbanian protein homolog
• Kuzbanian
• MADM
• MADMEC 3.4.24.81
• Mammalian disintegrin-metalloprotease

Background

ADAM10 (also known as Kuzbanian, mammalian disintegrin metalloprotease, myelin-associated metalloproteinase) is a member of the ADAM family that contains a disintegrin and metalloprotease-like domain (1, 2). Like other membrane-anchored ADAMs, ADAM10 consists of the following domains, pro with a cysteine switch and furin cleavage sequence, catalytic with the zinc-binding site and Met-turn expected for reprolysins, disintegrin-like, cysteine-rich, EGF-like, transmembrane, and cytoplasmic. ADAM10 is highly conserved, with 97% amino acid identity between mouse, rat, bovine and human and 45% identity between mouse and Drosophila. The active enzyme processes notch, notch ligand delta, and amyloid protein precursor at the alpha site, playing an important role in neurogenesis (3, 4). It also processes the 26 kDa membrane-anchored pro-tumor necrosis factor-alpha (TNF-alpha ) to the 17 kDa mature TNF-alpha (5). It cleaves myelin basic protein and type IV collagen (6, 7). ADAM10 is widely expressed in tissues and resides both on the cell surface and in the cell (8, 9).

1. Rooke, J. et al. (1996) Science 273:1227.
2. Pan, D. and Rubin, G.M. (1997) Cell 90:271.
3. Qi, H. et al. (1999) Science 283:91.
4. Lammich, S. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3922.
5. Rosendahl, M.S. et al. (1997) J. Biol. Chem. 272:24588.
6. Chantry, A. et al. (1989) J. Biol. Chem. 264:21603.
7. Millichip, M.I. et al. (1998) Biochem. Biophys. Res. Comm. 245:594.
8. Chantry, A. and Glynn, P. (1990) Biochem. J. 268:245.
9. Fahrenholz, F.S. et al. (2000) Ann. N.Y. Acad. Sci. 920:215.
   
   

Publications for ADAM10 (946-AD)(3)

Publications using 946-AD

• Burmeister, M;Fraunenstein, A;Kahms, M;Arends, L;Gerwien, H;Deshpande, T;Kuhlmann, T;Gross, CC;Naik, VN;Wiendl, H;Klingauf, J;Meissner, F;Sorokin, L; Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function Science advances 2023-07-21 [PMID: 37467333] (Bioassay, Mouse)
• A-Disintegrin and Metalloproteinase (ADAM) 17 Enzymatically Degrades Interferon-gamma Sci Rep, 2016-08-30;6(0):32259. 2016-08-30 [PMID: 27573075] (Bioassay, Mouse)
• Zhou BB, Peyton M, He B, Liu C, Girard L, Caudler E, Lo Y, Baribaud F, Mikami I, Reguart N, Yang G, Li Y, Yao W, Vaddi K, Gazdar AF, Friedman SM, Jablons DM, Newton RC, Fridman JS, Minna JD, Scherle PA Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell, 2006-07-01;10(1):39-50. 2006-07-01 [PMID: 16843264] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: Adam10
• Uniprot:
  • O35598()