Recombinant Mouse Hepsin Protein, CF Summary
Product Specifications
Gln44-Pro416 (Asp160Glu & Arg161Lys), 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.
9156-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate 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: 0.1 M Tris, 10 mM CaCl2, 0.15 M NaCl, 0.05% Brij-35, pH 8.0
- Assay Buffer: 50 mM Tris, pH 9.0
- Recombinant Mouse Hepsin (rmHepsin) (Catalog # 9156-SE)
- Substrate: BOC-Gln-Arg-Arg-AMC (Bachem, Catalog # I-1655), 5 mM stock in 50:50 DMSO:Methanol
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmHepsin to 100 µg/mL in Activation Buffer.
- Incubate at 37 °C for 24 hours.
- Dilute activated rmHepsin to 0.2 µg/mL in Assay Buffer.
- Dilute Substrate to 200 μM in Assay Buffer.
- Load into a plate 50 μL of 0.2 µg/mL rmHepsin, and start the reaction by adding 50 μL of 200 μM Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.
- Read plate at excitation and emission wavelengths of 380 nm and 460 nm, 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-Methyl Coumain (AMC) (Sigma, Catalog # A9891).
- rmHepsin: 0.010 μg
- Substrate: 100 μM
Reconstitution Calculator
Background: Hepsin
Hepsin, also known as TMPRSS1, is an approximately 50 kDa Type II membrane protein with an extracellular serine protease domain. It is primarily expressed in liver as a disulfide linked heterodimer of heavy and light chains (1, 2). Hepsin cleaves and activates Coagulation Factors VII, IX, and XII (3, 4), the pro forms of HGF, MSP, and uPA (4-8), and Prostasin/PRSS8 (9). It also cleaves EGF R and prevents receptor binding of EGF [9]. Its enzymatic activity is inhibited by HAI-1 and HAI-2 (4, 5, 8). Hepsin is overexpressed in various human tumors including prostate and breast (6, 10), and it promotes tumor progression and metastasis (11). It disrupts attachment of epithelial cells to the basement membrane by inducing the breakdown of desmosomal structures (6). Hepsin also binds to Protein X from hepatitis B virus and to pORF3 from hepatitis E virus (12, 13). Within the ECD, mouse Hepsin shares 89% and 98% amino acid (aa) sequence identity with human and rat Hepsin, respectively (2). An alternatively spliced isoform has a 20 aa deletion in the cytoplasmic and transmembrane regions (2).
- Tsuji, A. et al. (1991) J. Biol. Chem. 266:16948.
- Vu, T.-K.H. et al. (1997) J. Biol. Chem. 272:31315.
- Kazama, Y. et al. (1995) J. Biol. Chem. 270:66.
- Herter, S. et al. (2005) Biochem. J. 390:125.
- Kirchhofer, D. et al. (2005) FEBS Lett. 579:1945.
- Tervonen, T.A. et al. (2016) Oncogene 35:1832.
- Ganesan, R. et al. (2011) Mol. Cancer Res. 9:1175.
- Moran, P. et al. (2006) J. Biol. Chem. 281:30439.
- Chen, M. et al. (2010) Mol. Cell. Biochem. 337:259.
- Dhanasekaran, S.M. et al. (2001) Nature 412:822.
- Li, W. et al. (2009) Cancer Res. 69:8395.
- Zhang, M. et al. (2015) Oncotarget 7:7780.
- Wang, C. et al. (2014) Hepat. Mon. 14:e13902.
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Average Rating: 4 (Based on 1 Review)
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Reason for Rating: - pros: recombinant protein shows strong activity, can be used for many, many samples- cons: somewhat expensive for a small experiment (given than you have to purchase the substrate and the other reagents)
- added 50 uL of 50 uM of substrate in each well
- added 1, 2,3,4, or 5 ng in each well
