Recombinant Human Hepsin Protein, CF
Recombinant Human Hepsin Protein, CF Summary
Product Specifications
Arg45-Leu417 (Asp161Glu & Arg162Lys), 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.
4776-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 Human Hepsin (rhHepsin) (Catalog # 4776-SE)
- Fluorogenic Peptide 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 rhHepsin to 100 µg/mL in Activation Buffer.
- Incubate at 37 °C for 24 hours.
- Dilute activated rhHepsin to 0.2 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of the 0.2 ng/µL rhHepsin in a black well plate and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), 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 Coumarin (AMC) (Sigma, Catalog # A-9891).
Per Well:- rhHepsin: 0.010 µg
- Substrate: 100 µM
Reconstitution Calculator
Background: Hepsin
Hepsin, also known as TMPRSS1, is a Type II membrane protein with an extracellular serine protease domain (1). It is most highly expressed in liver, but is also present in many other tissues, notably lung, kidney, and skeletal muscle (2). A soluble form of Hepsin lacking the transmembrane domain has been identified (3). Hepsin is capable of activating Factor VII, and may initiate blood coagulation at the cell surface (4). Hepsin is overexpressed in various human tumors, including prostate (5), and is considered to be a biomarker for prostate cancer (6). Recombinant human Hepsin was expressed as a secreted, soluble protein lacking its cytosolic and transmembrane domains. The D161E and R162K mutations were introduced into the prosequence to improve expression of the recombinant human Hepsin.
- Leytus, S.P. et al. (1988) Biochemistry 27:1067.
- Tsuji, A. et al. (1991) J. Biol. Chem. 266:16948.
- Li, Y. et al. (2005) Biomed. Biochim. Acta 1681:157.
- Kazama, Y. et al. (1995) J. Biol. Chem. 270:66.
- Dhanasekaran, S.M. et al. (2001) Nature 412:822.
- Wu, Q. and Parry, G. (2007) Front. Biosci. 12:5052.
Citations for Recombinant Human Hepsin 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 - 5
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Anti-Tumor Functions of Prelatent Antithrombin on Glioblastoma Multiforme Cells
Authors: J Peñas-Mart, G Luengo-Gil, S Espín, N Bohdan, C Ortega-Sab, MC Ródenas, D Zaragoza-H, MJ López-Andr, C Plasencia, V Vicente, A Carmona-Ba, I Martínez-M
Biomedicines, 2021-05-07;9(5):.
Species: Human
Sample Types: Protein
Applications: Bioassay -
Structure-activity relationship studies of dipeptide-based hepsin inhibitors with Arg bioisosteres
Authors: H Kwon, H Ha, H Jeon, J Jang, SH Son, K Lee, SK Park, Y Byun
Bioorganic chemistry, 2020-11-28;0(0):104521.
Species: N/A
Sample Types: Peptide
Applications: Bioassay -
Design and synthesis of dye-conjugated hepsin inhibitors
Authors: K Kim, H Kwon, D Choi, T Lim, I Minn, SH Son, Y Byun
Bioorg. Chem., 2019-05-20;89(0):102990.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform.
Authors: Pant S, Mukonoweshuro A, Desai B, Ramjee M, Selway C, Tarver G, Wright A, Birchall K, Chapman T, Tervonen T, Klefstrom J
J Med Chem, 2018-05-14;61(10):4335-4347.
Applications: Enzyme Assay -
Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion.
Authors: Tervonen T, Belitskin D, Pant S, Englund J, Marques E, Ala-Hongisto H, Nevalaita L, Sihto H, Heikkila P, Leidenius M, Hewitson K, Ramachandra M, Moilanen A, Joensuu H, Kovanen P, Poso A, Klefstrom J
Oncogene, 2015-07-13;0(0):ePub.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
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AF4776 was used as the detection antibody in a sandwich ELISA with MAB4776 as the capture antibody and 4776-SE-010 as the calibrator.
Activated hepsin was used at a concentration of 0.03nM. Assay was performed in PBS. The hepsin inhibitor was from Pant et al., 2018 (J Med Chem. 2018 May 24;61(10):4335-4347. doi: 10.1021/acs.jmedchem.7b01698. Epub 2018 May 14.
Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform.)
Loaded 0.1ug, reduces SDS-PAGE on gradient gel, followed by silver staining
