Recombinant Mouse Cathepsin H Protein, CF

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Product Details

Summary
Reactivity MuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

Order Details

Recombinant Mouse Cathepsin H Protein, CF Summary

Details of Functionality
Measured by its ability to cleave the fluorogenic peptide substrate, Arg-7-amido-4-methylcoumarin (R-AMC). The specific activity is >400 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
Source
Mouse myeloma cell line, NS0-derived mouse Cathepsin H protein
Glu22-Val333, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Glu22
Structure / Form
Pro form
Protein/Peptide Type
Recombinant Enzymes
Gene
Ctsh
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
36 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
40 kDa, reducing conditions
Publications
Read Publication using
1013-CY in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in MES and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
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
  1. Dilute rmCathepsin H to 200 µg/mL in Activation Buffer.
  2. Dilute Thermolysin to 100 µg/mL in Activation Buffer.
  3. Mix equal volumes of 100 µg/mL Thermolysin and 200 µg/mL rmCathepsin H.
  4. Incubate at 37 °C for 1 hour.
  5. Stop reaction by adding an equal volume of 2 mM Phosphoramidon in Assay Buffer to reaction mixture.
  6. Incubate at room temperature for 10 minutes.
  7. 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.
  8. Incubate reaction at room temperature for 30 minutes.
  9. Dilute activated rmCathepsin H to 0.5 ng/µL in Assay Buffer.
  10. Dilute Substrate to 200 µM in Assay Buffer.
  11. 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.
  12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  13. 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:
  • rmCathepsin H: 0.025 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Cathepsin H Protein, CF

  • ACC-4
  • ACC-5
  • aleurain
  • cathepsin B3
  • cathepsin BA
  • Cathepsin H
  • CPSB
  • CTSH
  • DKFZp686B24257
  • EC 3.4.22
  • EC 3.4.22.16
  • MGC1519
  • minichain
  • N-benzoylarginine-beta-naphthylamide hydrolase

Background

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).

  1. Kirschke, H. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 1089, Academic Press, San Diego. 
  2. Lafuse, W.P. et al. (1995) J. Leukoc. Biol. 57:663.
  3. Soderstrom, M. et al. (1999) Biochim. Biophys. Acta 1446:35.
  4. Waghray, A. et al. (2002) J. Biol. Chem. 277:11533.
  5. Guncar, G. et al. (1998) Structure 6:51.
  6. Cimerman, N. et al. (2001) Clin. Chim. Acta 310:113.
  7. del Re, E.C. et al. (2000) Br. J. Cancer. 82:1317.

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Publications for Cathepsin H (1013-CY)(1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: EnzAct.


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Bioinformatics

Gene Symbol Ctsh
Entrez
Uniprot