Recombinant Human HAI-2A Protein, CF Summary
Product Specifications
Met1-Lys197, 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.
1106-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 at ambient temperature. 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-2A (rhHAI-2A) (Catalog # 1106-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 with Assay Buffer.
- Prepare a curve of rhHAI-2A (MW: 20539 Da) in Assay Buffer. Make the following serial dilutions: 1000, 100, 33.3, 16.7, 8.33, 4.17, 2.08, and 0.417 nM.
- Mix equal volumes of the rhHAI-2A curve dilutions and the diluted Trypsin. Include a control (in duplicate) containing Assay Buffer and the diluted Trypsin without any rhHAI-2A.
- Incubate reactions for 30 minutes at room temperature.
- After incubation, dilute the mixtures by 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) 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)
- Trypsin: 0.00125 µg
- rhHAI-2A curve: 50, 5, 1.67, 0.83, 0.417, 0.209, 0.104, and 0.0209 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: HAI-2A
Two alternatively spliced forms of HAI-2 have been found in human tissues (1). HAI-2A, the full-length molecule and also known as placental bikunin, is a major form expressed in human tissues. Encoded by the SPINT2 gene, HAI-2A consists of two Kunitz domains, and a C-terminal transmembrane domain (1‑5). Both Kunitz domains can function as inhibitors independent of each other. In addition to HGF activator and trysin, HAI-2A strongly inhibits plasmin, tissue and plasma kallikreins, and factor XIa. In comparison, HAI-2A is a weaker inhibitor of factor VIIa-tissue factor, factors IXa, Xa, and XIIa. Recombinant HAI-2A prolonged the clotting time in an activated partial thromboplastin time assay.
- Kataoka, H. et al. (2002) Biochem. Biophys. Res. Comm. 290:1096.
- Kawaguchi, T. et al. (1997) J. Biol. Chem. 272:27558.
- Marlor. C.W. et al. (1997) J. Biol. Chem. 272:12202.
- Muller-Pillasch, F. et al. (1998) Biochim. Biophys. Acta 1395:88.
- Delaria, K.A. et al. (1997) J. Biol. Chem. 272:12209.
Citations for Recombinant Human HAI-2A 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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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 -
Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling.
Authors: Zoratti G, Tanabe L, Varela F, Murray A, Bergum C, Colombo E, Lang J, Molinolo A, Leduc R, Marsault E, Boerner J, List K
Nat Commun, 2015-04-15;6(0):6776.
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Activation -
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.
Species: Human
Sample Types: N/A, Whole Cells
Applications: Bioassay, Degradation
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