Recombinant Human Neutrophil Elastase/ELA2 Protein, CF
Recombinant Human Neutrophil Elastase/ELA2 Protein, CF Summary
Product Specifications
Ser28-Asn252, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
9167-SE
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES and NaCl. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
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 Human Elastase/ELA2 (rhELA2) (Catalog # 9167-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 stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhELA2 to 50 µg/mL in Activation Buffer containing 50 µg/mL rmCathepsin C.
- Incubate for 2 hours at 37 °C to activate rhELA2.
- Dilute active rhELA2 to 1 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load into a plate 50 µL of 1 ng/µL rhELA2, 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.
- Read at excitation and emission wavelengths of 380 nm and 460 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 7-amino, 4-Methly Coumarin (Sigma, Catalog # A9891).
- rhELA2: 0.05 µg
- Substrate: 100 µM
Reconstitution Calculator
Background: Neutrophil Elastase/ELA2
Neutrophil Elastase (ELA2, ELANE), also known as HNE, is a chymotrypsin family serine protease that plays a key role in pathogen clearance (1-3). It is expressed by promyelocytes and stored in the intracellular azurophilic granules of polymorphonuclear leukocytes (PMN) (4). These granules fuse with phagosomes, enabling Neutrophil Elastase to participate in the digestion and killing of endocytosed microbes. The enzyme is released by activated neutrophils at sites of inflammation, and it can remain associated with the cell surface or function as a component of neutrophil extracellular nets (NETs) which trap and kill microbial pathogens (5, 6). It also can degrade multiple extracellular matrix proteins including Elastin and Fibronectin (5). In the lung, this activity contributes to pathology in emphysema, cystic fibrosis, and adult respiratory distress syndrome (ARDS) (1). Neutrophil Elastase can be inhibited by Serpin A1/alpha 1-Antitrypsin, SLPI, Serpin B1, and
Trappin-2/Elafin (7-11). Its activity in the lung is increased by exposure to tobacco smoke which inactivates Serpin A1 through methionine oxidation (12). Mature human Neutrophil Elastase shares 73% amino acid sequence identity with mouse and rat Neutrophil Elastase (13, 14). Multiple mutations in the human ELANE gene are causative of severe congenital and cyclic neutropenias (15).
- Korkmaz, B. et al. (2010) Pharmacol. Rev. 62:726.
- Stein, R.L. et al. (1987) Biochemistry 26:1301.
- Bachovchin, W.W. (1986) Biochemistry 25:7751.
- Garwicz, D. et al. (2005) Haematologica 90:38.
- Owen, C.A. et al. (1995) J. Cell Biol. 131:775.
- Stephan, A. and M. Fabri (2015) Exp. Dermatol. 24:161.
- Carrell, R.W. et al. (1982) Nature 298:329.
- Rice, W.G. and S.J. Weiss (1990) Science 249:178.
- Thompson, R.C. et al. (1986) Proc. Natl. Acad. Sci. USA 83:6692.
- Cooley, J. et al. (2001) Biochemistry 40:15762.
- Wiedow, O. et al. (1990) J. Biol. Chem. 265:14791.
- Taggart, C. et al. (2000) J. Biol. Chem. 275:27258.
- Sinha, S. et al. (1987) Proc. Natl. Acad. Sci. USA 84:2228.
- Okano, K. et al. (1987) J. Biochem. 102:13.
- Makaryan, V. et al. (2015) Curr. Opin. Hematol. 22:3.
Citations for Recombinant Human Neutrophil Elastase/ELA2 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.
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Citations: Showing 1 - 3
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Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation
Authors: P Cheung, S Schaffert, SE Chang, M Dvorak, M Donato, C Macaubas, MH Foecke, TM Li, L Zhang, JP Coan, GS Schulert, AA Grom, LA Henderson, PA Nigrovic, JE Elias, O Gozani, ED Mellins, P Khatri, PJ Utz, AJ Kuo
Nature Immunology, 2021-05-20;22(6):711-722.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
The Chymase Mouse Mast Cell Protease-4 Regulates Intestinal Cytokine Expression in Mature Adult Mice Infected with Giardiaintestinalis
Authors: Z Li, D Peirasmaki, S Svärd, M Åbrink
Cells, 2020-04-09;9(4):.
Species: Human
Sample Types: Protein
Applications: Bioassay -
Cathepsin G cleaves and activates IL-36? and promotes the inflammation of psoriasis
Authors: J Guo, J Tu, Y Hu, G Song, Z Yin
Drug Des Devel Ther, 2019-02-08;13(0):581-588.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
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