Recombinant Mouse Cathepsin H Protein, CF Summary
Product Specifications
Glu22-Val333, 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.
1013-CY
| Formulation | Supplied as a 0.2 μm filtered solution in MES and NaCl. |
| 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
- Activation Buffer: 50 mM MES, 10 mM CaCl2, 150 mM NaCl, pH 6.5
- Assay Buffer: 50 mM MES, pH 6.5
- Dithiothreitol (DTT) (Sigma, Catalog # D-0632), 1 M stock in deionized water
- Recombinant Mouse Cathepsin H (rmCathepsin H) (Catalog # 1013-CY)
- Bacterial Thermolysin (Catalog # 3097-ZN)
- Phosphoramidon (Catalog # EI006), 20 mM in methanol
- Substrate: Arg-7-amido-4-methylcoumarin (R-AMC) (Chem-Impex, Catalog # 5859), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmCathepsin H to 200 µg/mL in Activation Buffer.
- Dilute Thermolysin to 100 µg/mL in Activation Buffer.
- Mix equal volumes of 100 µg/mL Thermolysin and 200 µg/mL rmCathepsin H.
- Incubate at 37 °C for 1 hour.
- Stop reaction by adding an equal volume of 2 mM Phosphoramidon in Assay Buffer to reaction mixture.
- Incubate at room temperature for 10 minutes.
- Then add an equal volume of Assay Buffer containing 20 mM DTT to reaction mixture. The concentration of rmCathepsin H is now 25 ng/µL.
- Incubate reaction at room temperature for 30 minutes.
- Dilute activated rmCathepsin H to 0.5 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- In a plate load 50 µL of 0.5 ng/µL rmCathepsin H to wells, 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)
- rmCathepsin H: 0.025 µg
- Substrate: 100 µM
Background: Cathepsin H
Cathepsin H is a lysosomal cysteine protease of the papain family (1). It is synthesized as a precursor protein, consisting of a signal peptide (residues 1‑20), a propeptide (residues 21‑95), a mini chain (residues 96‑103), a heavy chain (residues 114‑290) and a light chain (residues 291‑333) (2, 3). A truncated form with a 12 amino acid deletion in the signal peptide region is secreted (4). Cathepsin H is the only known mono-aminopeptidase in the papain family (5). Cathepsin H expression is significantly increased in disease states such as in prostate tumors, sera of asthmatic patients, and mucosa of colorectal cancer patients (4, 6, 7).
- Kirschke, H. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 1089, Academic Press, San Diego.
- Lafuse, W.P. et al. (1995) J. Leukoc. Biol. 57:663.
- Soderstrom, M. et al. (1999) Biochim. Biophys. Acta 1446:35.
- Waghray, A. et al. (2002) J. Biol. Chem. 277:11533.
- Guncar, G. et al. (1998) Structure 6:51.
- Cimerman, N. et al. (2001) Clin. Chim. Acta 310:113.
- del Re, E.C. et al. (2000) Br. J. Cancer. 82:1317.
Citation for Recombinant Mouse Cathepsin H 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.
1 Citation: Showing 1 - 1
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Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.
Authors: Burster T, Marin-Esteban V, Boehm BO, Dunn S, Rotzschke O, Falk K, Weber E, Verhelst SH, Kalbacher H, Driessen C
Biochem. Pharmacol., 2007-08-02;74(10):1514-23.
Species: Human
Sample Types: Cell Lysates
Applications: Enzyme Assay
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