Recombinant Human Cathepsin H Protein, CF Summary
Product Specifications
Ala23-Val335, 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.
7516-CY
| Formulation | Supplied as a 0.2 μm filtered solution in Tris 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: 50 mM MES, 10 mM CaCl2, 150 mM NaCl, pH 6.5
- Assay Buffer: 50 mM MES, pH 6.5
- Recombinant Human Cathepsin H (rhCathepsin H) (Catalog # 7516-CY)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- Phosphoramidon (Tocris, Catalog # 6333), 20 mM stock in methanol
- Substrate: Arg-7-amino-4-methylcoumarin (R-AMC) (ChemImpex, 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 rhCathepsin 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 rhCathepsin H.
- Incubate at RT for 3 hours.
- Stop reaction by adding an equal volume of 2 mM Phosphoramidon in Assay Buffer to reaction mixture.
- Incubate at RT for 10 minutes.
- Add an equal volume of Assay Buffer containing 20 mM DTT to reaction mixture. The concentration of rhCathepsin H is now 25 µg/mL.
- Incubate reaction at RT for 5 minutes.
- Dilute activated rhCathepsin H to 1 µg/mL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- In a plate, load 50 µL of 1 µg/mL rhCathepsin 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-MethylCoumarin (AMC) (Sigma, Catalog # 9891).
- rhCathepsin H: 0.05 µg
- Substrate: 100 µM
Reconstitution Calculator
Background: Cathepsin H
Cathepsin H is a lysosomal cysteine protease of the papain family (1). It is synthesized as a precursor protein that is proteolytically processed to a mature form consisting of a light chain, a heavy chain, and a mini-chain (2). Cathepsin H is the only known mono-aminopeptidase in the papain family (3). Cathepsin H is primarily an aminopeptidase, but also functions as an endopeptidase (4). Cathepsin H is potently inhibited by cystatins A, B, and C and by ovocystatin. Cathepsin H expression is altered in disease states such as prostate and colorectal cancers (5).
- Kirshke, H. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett et al.) p. 1089, Academic Press, San Diego.
- Fuchs, R. et al. (1988) Biol. Chem. Hoppe-Seyler 369:469.
- Guncar, G. et al. (1998) Structure 310:113.
- Kirschke, H. et al. (1977) Acta Biol. Med. Ger. 6:51.
- del Re, E.C. et al. (2000) Br. J. Cancer 82:1317.
Citations for Recombinant Human 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.
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Citations: Showing 1 - 2
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Cancer-associated fibroblasts are key determinants of cancer cell invasion in the earliest stage of colorectal cancer
Authors: H Dang, TJ Harryvan, CY Liao, EHJ Danen, VNLN Spalburg, SM Kielbasa, H Mei, JJ Goeman, ES de Jonge-M, SGT Janson, JJ van der Re, S Crobach, JCH Hardwick, JJ Boonstra, NFCC de Miranda, LJAC Hawinkels
Cellular and Molecular Gastroenterology and Hepatology, 2023-04-20;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
Applications: Bioassay -
Suppression and resolution of autoimmune arthritis by rhesus ?-defensin-1, an immunomodulatory macrocyclic peptide
Authors: JB Schaal, DQ Tran, A Subramania, R Patel, T Laragione, KD Roberts, K Trinh, P Tongaonkar, PA Tran, D Minond, GB Fields, P Beringer, AJ Ouellette, PS Gulko, ME Selsted
PLoS ONE, 2017-11-16;12(11):e0187868.
Applications: Bioassay
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