Recombinant Human HAI-1 Protein, CF Summary
Product Specifications
Pro37-Glu449, 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.
1048-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris and NaCl. |
| 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 Human HAI-1 (rhHAI-1) (Catalog # 1048-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 in Assay Buffer.
- Prepare a curve of rhHAI-1 (MW: 47489 Da) in Assay Buffer. Make the following serial dilutions: 500, 200, 50, 25, 10, 5, 2, 0.5, and 0.05 nM.
- Mix equal volumes of the rhHAI-1 curve dilutions and the diluted Trypsin. Include a control (in duplicate) containing Assay Buffer and the diluted Trypsin.
- Incubate reactions for 1 hour at 37 °C.
After incubation, dilute the mixtures 5 fold in Assay Buffer. - Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the diluted incubated mixtures into a plate 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.
- Derive the 50% inhibiting concentration (IC50) of rhHAI-1 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for trypsin at each point may be determined 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
- rhHAI-1 curve: 25, 10, 2.5, 1.25, 0.5, 0.25, 0.1, 0.025, 0.0025 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: HAI-1
HAI-1 is a Kunitz-type serine protease inhibitor, identified as a strong inhibitor of HGF activator (HGFA) and matriptase (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, K. et al. (2002) J. Biol. Chem. 277:14053.
- Shimomura, T. et al. (1997) J. Biol. Chem. 272:6370.
- Kataoka, H. et al. (2000) J. Biol. Chem. 275:40453.
Citations for Recombinant Human 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.
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Citations: Showing 1 - 6
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Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy
Authors: R Szabo, TH Bugge
PLoS ONE, 2018-04-04;13(4):e0194660.
Applications: Bioassay -
Phosphorylation of the type II transmembrane serine protease, TMPRSS13 in Hepatocyte Growth Factor Activator Inhibitor-1 and 2-mediated cell surface localization
Authors: AS Murray, FA Varela, TE Hyland, AJ Schoenbeck, JM White, LM Tanabe, SV Todi, K List
J. Biol. Chem., 2017-07-14;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Matriptase initiates activation of epidermal pro-kallikrein and disease onset in a mouse model of Netherton syndrome.
Authors: Sales KU, Masedunskas A, Bey AL
Nat. Genet., 2010-07-25;42(8):676-83.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
Potent Inhibition and Global Co-localization Implicate the Transmembrane Kunitz-type Serine Protease Inhibitor Hepatocyte Growth Factor Activator Inhibitor-2 in the Regulation of Epithelial Matriptase Activity.
Authors: Szabo R, Hobson JP, List K, Molinolo A, Lin CY, Bugge TH
J. Biol. Chem., 2008-08-19;283(43):29495-504.
Applications: Degradation -
Pharmacoproteomics of a metalloproteinase hydroxamate inhibitor in breast cancer cells: dynamics of membrane type 1 matrix metalloproteinase-mediated membrane protein shedding.
Authors: Butler GS, Dean RA, Tam EM, Overall CM
Mol. Cell. Biol., 2008-05-27;28(15):4896-914.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5.
Authors: Cortesio CL, Jiang W
Arch. Biochem. Biophys., 2006-03-03;449(1):164-70.
Applications: Enzyme Assay
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