Recombinant Mouse Neutrophil Elastase/ELA2 Protein, CF

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

Recombinant Mouse Neutrophil Elastase/ELA2 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, MeOSuc-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (MeOSuc-AAPV-AMC). The specific activity is >500 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Neutrophil Elastase/ELA2 protein
  Ser27-Arg260, with a C-terminal 10-His tag
• Accession #: NP_056594
• N-terminal Sequence: Ser27
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Elane
• Purity: >95%, 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: 27 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: 34 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
  • Assay Buffer: 50 mM Tris, 1 M NaCl, 0.05% (w/v) Brij-35, pH 7.5
  • Recombinant Mouse Neutrophil Elastase/ELA2 (rmELA2) (Catalog # 4517-SE)
  • Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
  • Substrate: MeOSuc-Ala-Ala-Pro-Val-AMC (Bachem, Catalog # I-1270), 10 mM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmELA2 to 50 µg/mL in Activation Buffer containing 50 µg/mL rmCathepsin C.
  2. Incubate for 2 hours at 37 °C to activate rmELA2.
  3. Dilute active rmELA2 to 1 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer.
  5. Load into a plate 50 µL of 1 ng/µL rmELA2, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
  6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. 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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

  Per Well:
  • rmELA2: 0.05 µg
  • Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Mouse Neutrophil Elastase/ELA2 Protein, CF

• Bone marrow serine protease
• EC 3.4.21
• EC 3.4.21.37
• ELA2
• ELA2granulocyte-derived elastase
• ELANE
• elastase 2, neutrophil
• elastase, neutrophil expressed
• Elastase-2
• GE
• HLEelastase-2
• HNE
• Human leukocyte elastase
• Leukocyte Elastase
• Medullasin
• NE
• Neutrophil Elastase
• PMN elastase
• PMN-E
• polymorphonuclear elastase
• SCN1

Background

Neutrophil Elastase (ELA2), also known as polymorphonuclear leukocyte elastase, is a serine protease belonging to the chymotrypsin family. Located primarily in the azurophil granules of polymorphonuclear leukocytes, ELA2 function is the degradation of many extracellular matrix macromolecules (1, 2). alpha -1 Antitrypsin (Serpin A1) and secretory leukocyte protease inhibitor (SLPI) have been shown to inhibit ELA2 activity (3). This protein may be involved in lung emphysema, cystic fibrosis, the adult respiratory distress syndrome (ARDS), rheumatoid arthritis, tumor invasion and infectious diseases (1). The purified rmELA2 does not contain the last five amino acid residues of the deduced amino acid sequence (NP_056594).

1. Bieth, J.G. (2004) In Handbook of Proteolytic Enzymes. Barrett, A.J., Rawlings, N.D., & Woessner, J.F. eds. pp. 1517.
2. Owen, C.A. et al. (1995) J. Cell Biol. 131:775.
3. Wiesner, O. et al. (2005) FEBS letters 579:5305.

Publications for Neutrophil Elastase/ELA2 (4517-SE)(5)

Publications using 4517-SE

• Chen, D;Mirski, MA;Caton, ER;Kiser, KM;Haddaway, CR;Cetta, MS;Chen, S;Bryden, WA;McLoughlin, M; A Breath-Based In Vitro Diagnostics for Lower Respiratory Tract Infection medRxiv : the preprint server for health sciences 2023-09-18 [PMID: 37790344] (Bioassay, Mouse)
• L Zhou, R Gao, H Hong, X Li, J Yang, W Shen, Z Wang, J Yang Emodin inhibiting neutrophil elastase-induced epithelial-mesenchymal transition through Notch1 signalling in alveolar epithelial cells J. Cell. Mol. Med., 2020-09-15;0(0):. 2020-09-15 [PMID: 32935466] (Bioassay, Rat)
• Raymond W, Xu X, Nimishakavi S, Le C, McDonald D, Caughey G Regulation of hepatocyte growth factor in mice with pneumonia by peptidases and trans-alveolar flux. PLoS ONE, 2015-05-04;10(5):e0125797. 2015-05-04 [PMID: 25938594] (Bioassay, Mouse)
• Vicuna L, Strochlic D, Latremoliere A, Bali K, Simonetti M, Husainie D, Prokosch S, Riva P, Griffin R, Njoo C, Gehrig S, Mall M, Arnold B, Devor M, Woolf C, Liberles S, Costigan M, Kuner R The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase. Nat Med, 2015-04-27;21(5):518-23. 2015-04-27 [PMID: 25915831] (Bioassay, Mouse)
• Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase. Nat Med, 2012-09-01;18(9):1407-12. 2012-09-01 [PMID: 22863787] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: Elane
• Uniprot:
  • NP_056594()