Recombinant Human ADAM12 Protein, CF

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

Recombinant Human ADAM12 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave IGFBP-3.
• Source: Chinese Hamster Ovary cell line, CHO-derived human ADAM12 protein
  Arg29-Ser513, with an N-terminal signal peptide and a C-terminal 6-His tag
• Accession #: AAC08702
• N-terminal Sequence: Arg29
• Structure / Form: Pro & Mature forms
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ADAM12
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 55 kDa (Pro) & 34 kDa (Mature).
  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: 25-30 kDa, 50-55 kDa & 68-72 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 Sodium Acetate, CaCl₂ and NaCl.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 1 mM CaCl₂, 0.5% Brij-35, pH 9.0
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5
  • Recombinant Human ADAM12 (rhADAM12) (Catalog # 4416-AD)
  • Recombinant Human Furin (rhFurin) (Catalog # 1503-SE)
  • Recombinant Human IGFBP‑3 (rhIGFBP‑3) (Catalog # 675-B3)
  • SDS-PAGE and/or Western blot
  1. Dilute rhADAM12 to 200 µg/mL in Activation Buffer.
  2. Dilute rhFurin to 10 µg/mL in Activation Buffer.
  3. Mix 25 µL of rhADAM12 and 25 µL of rhFurin in a reaction tube.
  4. Incubate the mixture for 1 hour at 37 °C.
  5. Reconstitute or dilute rhIGFBP-3 to 200 µg/mL in Assay Buffer (do not follow reconstitution directions given by the 675-B3 product insert).
  6. Add 25 µL of rhIGFBP-3 dilution, 10 µL of activated rhADAM12, and 15 µL of Assay Buffer to a tube.
  7. Incubate reaction at 37 °C for 16 hours.
  8. As controls combine 25 µL of rhIGFBP-3 solution and 25 µL of Assay Buffer in each of two tubes. Incubate one tube at 37 °C and the other at -20 °C.
  9. Stop the reaction by adding SDS-PAGE sample buffer.
  10. Analyze the cleavage by SDS-PAGE followed by protein staining and/or Western blot.
  Per Reaction:
  • rhADAM12: 20 µg/mL
  • rhIGFBP-3: 100 µg/mL

Notes

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

Alternate Names for Recombinant Human ADAM12 Protein, CF

• a disintegrin and metalloproteinase domain 12 (meltrin alpha)
• ADAM 12
• ADAM metallopeptidase domain 12
• ADAM12
• disintegrin and metalloproteinase domain-containing protein 12
• EC 3.4.24
• EC 3.4.24.58
• MCMP
• MCMPMltna
• Meltrin alpha
• meltrin-alpha
• MLTNA
• MLTNEC 3.4.24.-

Background

ADAM12, also known as meltrin-alpha , is a member of the ADAM family with metalloprotease activity (1). It consists of a propeptide, metalloproteinase, disintegrin, cysteine-rich, and EGF-like domains, a transmembrane segment, and a cytoplasmic tail with SH3 binding motifs. Human ADAM12 exists in two alternatively spliced forms: the prototype transmembrane form and a shorter secreted form lacking the transmembrane domain and the cytoplasmic tail. The secreted form has a 34 amino acid substitution in place of the transmembrane and cytoplasmic regions. In mouse, only the transmembrane form has been observed. The propeptide, which is cleaved in the Golgi by furin-like proprotein convertases, is retained in a noncovalent complex after ADAM12 secretion (2). Thus, the pro domain may function as an inhibitor of the proteolytic activity or play another unknown function. The known physiological substrates of ADAM12 are HBEGF in the heart (3) and IGFBP-3 and ‑5 in placental serum (4). Its proteolytic activity is inhibited by the tissue inhibitor of metalloproteinase-3 (Recombinant Human TIMP‑3, (Catalog # 973-TM) and alpha ‑2‑macroglobulin. It also mediates cell-cell adhesion by interacting with integrins and syndecans as well as with additional unidentified molecules (4). ADAM12 may be a promising marker in prenatal diagnostics and breast cancer (5, 6). The recombinant ADAM12 contains the pro, metalloproteinase, and disintegrin domains. In addition to TIMP-3, the activity can also be inhibited by 5 mM 1,10-phenanthroline.

1. Wewer, U.M. (2004) in Handbook of Proteolytic Enzymes, Barrett, A. J. et al. eds. pp 724.
2. Wewer, U.M. et al. (2006) J. Biol. Chem. 281:9481.
3. Asakura, M. et al. (2002) Nat. Med. 8:35.
4. Loechel, F. et al. (2000) Biochem. Biophys. Res. Comm. 278:511.
5. Laigaard, J. et al. (2006) Prenat Diagn. 26:973.
6. Roopali, R. et al. (2004) J. Biol. Chem. 279:51323.

Publications for ADAM12 (4416-AD)(2)

Publications using 4416-AD

• H Kim, Y Fu, HJ Hong, SG Lee, DS Lee, HM Kim Structural basis for assembly and disassembly of the IGF/IGFBP/ALS ternary complex Nature Communications, 2022-07-30;13(1):4434. 2022-07-30 [PMID: 35907924] (Bioassay, Human, N/A)
• R Roy, A Dagher, C Butterfiel, MA Moses ADAM12 is a Novel Regulator of Tumor Angiogenesis via STAT3 Signaling Mol. Cancer Res., 2017-08-01;0(0):. 2017-08-01 [PMID: 28765266] (Bioassay, In Vivo, Human, Mouse)

Bioinformatics

• Gene Symbol: ADAM12
• Uniprot:
  • AAC08702()