Three alternatively spliced forms of HAI-2 have been found in mouse tissues (1). HAI-2A, the full-length molecule, consists of two Kunitz domains and a C-terminal transmembrane domain. HAI-2A is a major form expressed in human tissues, but a minor form in mouse tissues. HAI-2B, with residues 37‑93 spliced out and thus lack of the first Kunitz domain, is a major form in mouse tissues. HAI-2B is an effective inhibitor of both HGF activator and trypsin (2).
Recombinant Mouse HAI-2B Protein, CF
R&D Systems | Catalog # 1107-PI
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse HAI-2B Protein (1107-PI)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
NS0
Accession Number
Applications
Inhibition Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived mouse HAI-2B protein
Arg30-Lys140, with a C-terminal 10-His tag
Arg30-Lys140, with a C-terminal 10-His tag
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Arg30
Predicted Molecular Mass
14 kDa
SDS-PAGE
23 kDa, reducing conditions
Activity
Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).
The IC50 value is <2 nM, as measured under the described conditions.
Formulation, Preparation, and Storage
1107-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in PBS. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile, deionized water.
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| 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.
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Calculators
Background: HAI-2B
References
- Itoh, H. et al. (1999) Biochem. Biophys. Res. Comm. 255:740.
- Kataoka, H. et al. (2002) Biochem. Biophys. Res. Comm. 290:1096.
Long Name
HGF Activator Inhibitor Type 2B
Alternate Names
HAI2B
Entrez Gene IDs
20733 (Mouse)
Gene Symbol
SPINT2
UniProt
Additional HAI-2B Products
Product Documents for Recombinant Mouse HAI-2B Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse HAI-2B Protein, CF
For research use only
Related Research Areas
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Protocols
View specific protocols for Recombinant Mouse HAI-2B Protein, CF (1107-PI):
Materials
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse HAI-2B (rmHAI-2B) (Catalog # 1107-PI)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002), 2 mM stock in DMSO
- 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 rmHAI-2B (MW: 14,281 Da) in Assay Buffer. Make the following serial dilutions: 1000, 200, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.391 nM.
- Mix equal volumes of the rmHAI-2A curve dilutions and 0.25 µg/mL Typsin. Include a control (in duplicate) containing Assay Buffer and 0.25 µg/mL Typsin.
- Incubate reactions for 30 minutes at room temperature.
- 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% inhibition concentration (IC50) value for rmHAI-2B by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity of 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
- rmHAI-2B curve: 50, 10, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.0195 nM
- Substrate: 10 µM