Recombinant Mouse HAI-1 Protein, CF Summary
Product Specifications
Glu30-Glu443 (Cys325Ser), 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.
1141-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris, NaCl and CaCl2. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile, deionized water. |
| Shipping | The product is shipped with polar packs. 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
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse HAI-1 (rmHAI-1) (Catalog # 1141-PI)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute Trypsin to 0.25 µg/mL with Assay Buffer.
- Prepare a curve of rmHAI-1 (MW: 47792 Da) in Assay Buffer. Make the following serial dilutions: 100, 40, 20, 12, 8, 4, 2 and 1 nM.
- Mix equal volumes of the rmHAI-1 curve dilutions and the diluted Trypsin. Include a Trypsin control (in duplicate) containing Assay Buffer and the diluted Trypsin without any rmHAI-1.
- Incubate reactions for 60 minutes at 37 °C.
- After incubation, dilute the mixtures five-fold in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load into plate 50 µL of the diluted incubated mixtures, and start the reaction by adding 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity for each point using the following formula (if needed):
|
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:- Trypsin: 0.00125 µg
- rmHAI-1: 5 nM, 2 nM, 1 nM, 0.6 nM, 0.4 nM, 0.2 nM, 0.1 nM, 0.05 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: HAI-1
Hepatocyte growth factor activator inhibitor-1 (HAI-1) encoded by the Spint1 gene is a Kunitz-type serine protease inhibitor, identified as a strong inhibitor of HGF activator (HGFA) and matripase (1). The membrane-anchored HAI-1 consists of two Kunitz domains, a LDL-receptor-like domain, and a C-terminal transmembrane domain (2). Two soluble forms are generated by ectodomain shedding, one with a single Kunitz domain and the other with two Kunitz domains. HAI-1 is not only an inhibitor but also a specific receptor of active HGFA, acting as a reservoir of this enzyme on the cell surface (3). The shedding of HAI-1 and HGFA/HAI-1 complex is enhanced by treatment with phorbol 12-myristate, 13-acetate or IL-1 beta. The regulated shedding is completely inhibited by a synthetic zinc metalloprotease inhibitor (3).
- Denda, et al. (2002) J. Biol. Chem. 277:14053.
- Shimomura, et al. (1997) J. Biol. Chem. 272:6370.
- Kataoka, et al. (2000) J. Biol. Chem. 275:40453.
Citation for Recombinant Mouse HAI-1 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.
1 Citation: Showing 1 - 1
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Imbalance in the pro-hepatocyte growth factor activation system in bleomycin-induced lung fibrosis in mice.
Authors: Phin S, Marchand-Adam S, Fabre A, Marchal-Somme J, Bantsimba-Malanda C, Kataoka H, Soler P, Crestani B
Am. J. Respir. Cell Mol. Biol., 2009-05-15;42(3):286-93.
Applications: Western Blot
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